Compositions and methods of treating cancer harboring pikc3a mutations

ABSTRACT

A method of treating cancer cells having mutated PIK3CA gene or protein of a subject in need thereof includes administering to the subject a therapeutically effective amount of an inhibitor of one or more enzymes of the glutamine metabolism pathway.

RELATED APPLICATION

This application claims priority from U.S. Provisional Application No.61/992,541, filed May 13, 2014, the subject matter of which isincorporated herein by reference in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under Grant No. CA160060awarded by The National Institutes of Health. The United Statesgovernment has certain rights to the invention.

BACKGROUND

The “Warburg effect” and “glutamine dependency” are two of the mostwell-known metabolic reprogramming events that occur in cancer cells anddistinguish them from many types of normal cells. Normally, glucose isconverted to acetyl-CoA, which enters the tricarboxylic acid (TCA) cycleand undergoes oxidative phosphorylation in mitochondria. However, cancercells convert glucose to lactate even in the presence of oxygen(“Warburg effect”). It was previously thought that the Warburg effectwas caused by impaired mitochondrial function in cancer cells. However,recent studies have demonstrated that most cancer cells retainfunctional mitochondria. Instead of using glucose, most cancer cellsutilize glutamine to replenish the TCA cycle. As illustrated in FIG. 1,to enter the TCA cycle, glutamine is first deaminated by glutaminases(GLSs) to generate glutamate. Glutamate is then converted toα-ketoglutarate (α-KG) to replenish the TCA cycle. Three groups ofenzymes convert glutamate to α-KG: (1) glutamate pyruvate transaminases(GPTs), (2) glutamate oxaloacetate transaminases (GOTs) and (3)glutamate dehydrogenases (GLUDs). Glutamine metabolites are utilized toproduce ATP and synthesize macromolecules, thereby promoting tumorgrowth. It has long been known that most cancer cells are dependent onglutamine. Although glutamine is a non-essential amino acid, it isnevertheless a required supplement for culturing cancer cells.

Many oncogenes impact glutamine metabolism. Myc overexpression affectsglutamine levels by inducing the transcription of GLS1 and the glutaminetransporter SLC1A5 (aka ASCT2). In contrast, SLC1A5 expression isrepressed by the Rb tumor suppressor, whereas GLS2 was identified as atranscriptional target of p53. In addition, it has been shown that p53represses the expression of malic enzymes ME1 and ME2, therebyregulating glutamine-dependent NADPH production. A recent study showedthat loss of tumor suppressor VHL renders renal cell carcinomassensitive to glutamine deprivation through HIF-induced metabolicreprograming. Moreover, K-ras up-regulates the aminotransferase GOT1.Though all of these mechanisms impact the production or degradation ofglutamine or its metabolites, the reasons that many cancer cells aredependent on glutamine are still unknown or being actively debated.

PIK3CA encodes the catalytic subunit of phosphatidylinositol 3-kinase α(PI3Kα), which plays a key role in regulating cell proliferation,survival and motility. PIK3α consists of a catalytic subunit p110α andone of several regulatory subunits (a major one being p85α). Upon growthfactor stimulation, p85 is recruited to phosphorylated receptor proteinkinases and adaptor proteins, thereby activating PI3Kα. Activated PI3Kαconverts phosphatidylinositol-4,5-biophosphate (PIP2) tophosphatidylinositol-3,4,5-triphosphate (PIP3). The second message PIP3then activates PDK1 and AKT signaling downstream. PIK3CA is mutated in awide variety of human cancers.

SUMMARY

Embodiments described herein relate to methods of determining thesusceptibility, resistance, and/or sensitivity of cancer cells,precancerous cells or benign tumor cells in a subject to the treatmentwith an inhibitor of one more enzymes of the glutamine metabolismpathway, such as inhibitors of glutaminase and/or inhibitors ofaminotransferase (e.g., glutamate pyruvate transaminase, aspirateaminotransferase, and glutamate dehydrogenase).

In some embodiments, the method includes obtaining a sample of thecancer cells, the precancerous cells or the benign tumor cells from thesubject, assaying the cells in the sample for the presence of a mutatedPIK3CA gene or a mutant form of PIK3CA protein or a biologically activefragment thereof, and determining that the subject should be treatedwith the inhibitor if the cancer cells have the mutated PIK3CA gene orthe mutant form of PIK3CA protein.

In other embodiments, the method includes obtaining a sample of thecancer cells, the precancerous cells or the benign tumor cells from thesubject, measuring the level of GPT2 expression in the cancer cells,comparing the measured level of GPT2 expression in the cancer cells to acontrol level, and identifying the cancer is more susceptible totreatment with the inhibitor if there is an increase in the measuredlevels of GPT2 expression in the cancer cells compared to a controllevel.

In the above methods, the cancer cells and the precancerous cells areobtained from a tumor or a biological sample from the subject such astumor biopsy or a biological sample comprising urine, blood,cerebrospinal fluid, sputum, serum, stool or bone marrow. In anembodiment, a DNA or RNA hybridization assay is used to detect thePIK3CA DNA or RNA in the sample. In other embodiments, a DNA or RNAhybridization assay is used to detect the GTP2 levels in the sample.

The cancer to be treated, for example, includes lung cancer, digestiveand gastrointestinal cancers, gastrointestinal stromal tumors,gastrointestinal carcinoid tumors, colon cancer, rectal cancer, analcancer, bile duct cancer, small intestine cancer, and stomach (gastric)cancer, esophageal cancer, gall bladder cancer, liver cancer, pancreaticcancer, appendix cancer, breast cancer, ovarian cancer, renal cancer,cancer of the central nervous system, skin cancer, lymphomas,choriocarcinomas, head and neck cancers, osteogenic sarcomas, and bloodcancers.

Others embodiment described herein relate to a method for treating asubject having cancer, precancerous cells, or a benign tumor that has orharbors a mutated PIK3CA gene or mutant PIK3CA protein, by administeringto the subject a therapeutically effective amount of one more inhibitorof enzymes of the glutamine metabolism pathway, such as inhibitors ofglutaminase and/or inhibitors of aminotransferase. In some embodiments,the inhibitor can be an aminotransferase inhibitor, such asaminooxyacetate (AOA) or epigallocatechin gallate (EGCG). In otherembodiments, the inhibitor can be a glutaminase inhibitor such asbis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide or CB-839(Calithera Bioscience, San Francisco, Calif.). In some embodiments, theglutaminase inhibitor comprises CB-839 or a pharmaceutically acceptablesalt thereof. In other embodiments, the glutaminase inhibitor has theformula:

or a pharmaceutically acceptable salt thereof.

In other embodiments, the cancer is colorectal cancer.

In an embodiment, the therapeutically effective amount of the inhibitorcan be from about 0.1 mg/day to about 150 mg/day.

In the method of treatment, the inhibitor(s) can be administered orally,by injection, parenterally, by inhalation spray, topically, rectally,nasally, buccally, vaginally or via an implanted reservoir. In anotherembodiment, the inhibitor(s) can be administered locally to the site ofthe cancer or benign tumor. In an embodiment, the aminotransferaseinhibitor is aminooxyacetate (AOA), and the glutaminase inhibitor isbis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide or CB-839(Calithera Bioscience, San Francisco, Calif.).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the initial steps of glutaminemetabolism.

FIGS. 2(A-E) illustrate plots, graphs, and immunoblots showing PIK3CAmutant colorectal cancer cells (CRC) cells are more sensitive toglutamine deprivation. (A) Allele configuration of colorectal cancerlines with either the PIK3CA WT or mutant allele knocked out. (B) PIK3CAmutant clones are more sensitive to glutamine, but not glucose,deprivation. Cells of the indicated genotypes were grown in culture andcell numbers were counted under the following conditions: normal mediumin the presence of both glucose and glutamine (normal), medium withoutglutamine (-Gln) and medium without glucose (-Glc). (C-D) Glutaminedeprivation induces more apoptosis in PIK3CA mutant clones. Cells of theindicated genotypes were grown with or without 2 mM glutamine for 72hours. Cell apoptosis was measured by profiling sub-G1 cells and cleavedPARP. (E) Glutamine deprivation induces more apoptosis in PIK3CA mutantCRC cell line. The indicated CRC cell lines were grown without glutaminefor 72 hours. Cell apoptosis was measured by profiling sub-G1 cells andcleaved PARP. PIK3CA mutant cell lines: RKO and HT29; WT PIK3CA celllines: LoVo and SW480.

FIGS. 3(A-F) illustrate immunoblots and graphs showing the up-regulationof GPT2 by PIK3CA mutations renders CRC dependent on glutamine. (A) GPT2expression levels are up-regulated in PIK3CA mutant clones. RT-PCRanalyses of the indicated genes in the HCT116 and DLD1 CRC clones. (B)Western blot analyses of GPT2 in the PIK3CA mutant and WT clones. (C)GPT2 expression levels are higher in PIK3CA mutant CRC specimens.qRT-PCR analyses of GPT2 in tumors with no mutations in the PIK3CApathway including PIK3CA, PTEN, PDK1 AKTs and IRS (n=10) and tumors withPIK3CA mutations (n=10). Data are plotted as Whiskers (Min to Max).p<0.05, t test. (D) Knockdown of GPT2 makes PIK3CA mutant cellsresistant to glutamine deprivation GPT2 was knocked down with twoindependent shRNAs in the HCT116 PIK3CA mutant clone. Stable pools weregrown with or without glutamine for three days. Relative survival=(cellnumber in absence of Gln)/(cell number with Gln)×100%. (E) Knockdown ofGPT2 in PIK3CA WT clone does not alter its sensitivity to glutaminedeprivation. (F) Overexpression (OE) of GPT2 in PIK3CA WT cells rendersthem more sensitive to glutamine deprivation. The HCT116 PIK3CA WT clonewas transfected with a Flag-tagged GPT2 expression vector. Two stableclones that express Flag-GPT2 were grown with or without glutamine. Dataare presented as mean+SEM of three independent cultures. * p<0.05; **p<0.01 t test.

FIGS. 4(A-D) illustrate plots showing aminooxyacetate (AOA) inhibitsxenograft tumor growth of PIK3CA mutant CRCs but not PIK3CA WT CRCs.(A-B) AOA inhibits growth of xenograft formed by PIK3CA mutant clonesbut not PIK3CA WT clones. (A) Clones derived from HCT116 cells; (B)Clones derived from DLD1. (C) AOA inhibits growth of xenograft tumorsformed by four CRC cell lines harboring PIK3CA mutations. (D) AOA doesnot inhibit growth of xenograft tumors formed by two CRC cell lines withWT PIK3CA. N=5 mice in each experimental group. Data are presented asmean±SEM. For HCT116 and DLD1 PIK3CA mutant clones, HCT116, DLD1, RKOand HT29, AOA treatment significantly inhibits xenograft tumor growth.*** p<0.0001, two-way ANOVA analysis.

FIGS. 5(A-H) illustrate immunoblots and graphs showing ATF4 activatesGPT2 transcription. (A-B) ATF4 protein levels correlate with that ofGPT2 in PIK3CA WT and mutant clones. Western blot of lysates of culturedcells (A) or lysates of the xenograft tumors form by the HCT116 clones(B). (C) Overexpression of ATF4 in the HCT116 PIK3CA WT clone increasesGPT2 protein levels. (D) Knockdown of ATF4 in the HCT116 PIK3CA mutantclone decreases GPT2 transcription. (E) Knockdown of ATF4 in the HCT116PIK3CA mutant clone renders the cells less sensitive to glutaminedeprivation. (F) Knockdown of ATF4 in the HCT116 PIK3CA mutant clonedecreases transcription activity of a GPT2 promoter reporter. (G) Twoputative ATF4 binding sites in the GPT2 promoter and mutant sequencesthat abolish ATF4 binding. (H) ATF4 binding site mutants reducestranscriptional activity of GPT2 promoter reporter. Data are presentedas mean+SEM of three independent experiments. * p<0.05; *** p<0.001.

FIGS. 6(A-I) illustrate immunoblots, a schematic drawing, and graphshowing he p110α-PDK1-RSK2 signaling axis regulates ATF4 proteinstability. (A-B) ATF4 ubiquitnation levels are higher in the HCT116 WTclone than the mutant clone. Ubiquitination of endogenous ATF4 (A).Ubiquitination of ectopically expressed Flag-tagged ATF4 and HA-taggedubiquitin (B). (C) Inhibitors of PI3K, PDK1 and RSK2 reduce ATF4 proteinlevels in the HCT116 mutant clone. (D) Schematics of the p110α signalingpathway that regulates ATF4 protein stability. (E) Overexpression ofoncogenic p110α mutants in the HCT116 WT clone increases protein levelsof ATF4 and GPT2. (F) Kinase-dead mutation on top of p110α E545Kmutation reduces protein levels of ATF4 and GPT2 in the DLD1 PIK3CAmutant clone. (G) Kinase-dead mutation renders DLD1 PIK3CA mutant cloneless sensitive to glutamine deprivation. Data are presented as mean+SEMof three independent cultures. ** p<0.01. (H) Knockdown of PDK1 by twoindependent siRNAs reduce ATF4 and GPT2 protein levels in the HCT116PIK3CA mutant clone. (I) Knockdown of RSK2 by two independent siRNAsreduce ATF4 and GPT2 protein levels in the HCT116 PIK3CA mutant clone.

FIGS. 7(A-G) illustrate immunoblots showing phosphorylation of ATF4 5245by RSK2 enhances its binding to USP8 and protects ATF4 fromubiquitin-mediated degradation. (A) Knockdown of RSK2 in the HCT116PIK3CA mutant clone reduces pS245 ATF4. (B) Levels of pS245 ATF4 arehigher in the HCT116 PIK3CA mutant clone than the WT clone. (C) The ATF4S245A mutant is less stable than the WT protein. The indicatedconstructs were expressed in the HCT116 PIK3CA mutant clone and celllysates were blotted with the indicated antibodies. (D) Ubiquitinationlevels of ATF4 S245A mutant are higher than that of WT protein. (E) TheATF4 S245A mutant binds to less USP8 than the WT protein. (F) Knockdownof USP8 in the HCT116 PIK3CA mutant clone reduces the ATF4 proteinlevels. (G) Knockdown of USP8 increases the levels of ATF4ubiquitination.

FIGS. 8(A-D) illustrate graphs and schematic drawing showing metabolicprofiling of PIK3CA WT and mutant clones. (A) [¹³C₅-]Glutamine tracingof the TCA cycle intermediates in HCT116 WT and mutant (mut) clones.(B-C) Relative levels of ATP and NADH in the HCT116 WT and mutant cloneswith or without glutamine. (B) ATP; (C) NADH. (D) α-KG rescues theHCT116 mutant clone from cell death caused by glutamine (Gln)deprivation. Data are presented as mean+SEM of three independentcultures. * p<0.05; ** p<0.01.

FIGS. 9(A-C) illustrate graphs and an immunoblot showing aminooxyacetate(AOA) inhibits PIK3CA mutant tumor growth. (A) IC₅₀ of AOA in HCT116 andDLD1 PIK3CA WT and mutant clones. (B) Body weights of the mice withxenograft established from the indicated CRC cells before and after AOAtreatment. N=5 mice in each group. (C) Western blot analyses of GPT2protein levels in the indicated cell lines.

FIGS. 10(A-B) illustrate plots showing AOA synergizes with 5-FU toinhibit growth of HCT116 xenograft tumors.

FIGS. 11(A-C) illustrate plots and graph showing (A) enzyme kinetics ofGPT2. Recombinant GPT2 was mixed with α-KG and alanine. The productpyruvate was detected by a colorimetric assay. (B) IC₅₀ of AOA to GPT2.(C) Relative amounts of α-KG. Xenograft tumors were treated with 10mg/kg of AOA or vehicle. α-KG in a untreated tumor was set as 100%.

FIGS. 12(A-B) illustrate images and a plot showing the combination ofCB-839 and 5-FU shrinks HCT116 xenograft tumors.

FIGS. 13(A-B) illustrate graphs showing PIK3CA mutations render cancercells sensitive to EGCG and BPTES.

DETAILED DESCRIPTION

For convenience, certain terms employed in the specification, examples,and appended claims are collected here. Unless defined otherwise, alltechnical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a substituent”includes a single substituent as well as two or more substituents thatmay be the same or different, reference to “a compound” encompasses acombination or mixture of different compounds as well as a singlecompound, reference to “a pharmaceutically acceptable carrier” includestwo or more such carriers as well as a single carrier, and the like.

The term “agent” and “drug” are used herein to mean chemical compounds,mixtures of chemical compounds, biological macromolecules, or extractsmade from biological materials, such as bacteria, plants, fungi, oranimal particularly mammalian) cells or tissues that are suspected ofhaving therapeutic properties. The agent or drug may be purified,substantially purified, or partially purified.

The term “biological sample” or “sample” as used herein includes anybiological specimen obtained from a subject. Frequently, the sample willbe a “clinical sample”, i.e., a sample derived from a patient. Suchsamples include, but are not limited to, bodily fluids which may containcancer cells, e.g., blood; tissue or fine needle biopsy samples, lungtissue; and archival samples with known diagnosis, treatment and/oroutcome history. Biological samples may also include sections of tissuesor cells, such as frozen sections taken from histological purposes. Theterm biological sample also encompasses any material derived byprocessing the biological sample. Derived materials include, but are notlimited to, cells (or their progeny) isolated from the sample, proteinsor nucleic acid molecules extracted from the sample. Processing of thebiological sample may involve one or more of, filtration, distillation,extraction, concentration, inactivation of interfering components,addition of reagents, and the like. In some embodiments, the sample iswhole blood or a fractional component thereof such as plasma, serum, ora cell pellet. In certain embodiments, the sample is obtained byisolating circulating cells of a solid tumor from a whole blood cellpellet using any technique known in the art. As used herein, the term“circulating cancer cells” comprises cells that have either metastasizedor micro metastasized from a solid tumor and includes circulating tumorcells, and cancer stem cells. In other embodiments, the sample is aformalin fixed paraffin embedded (FFPE) tumor tissue sample, e.g., froma solid tumor.

The term “control sample” refers to one or more biological samplesisolated from an individual or group of individuals that are normal(i.e., healthy).

The term “cancer” is intended to include any member of a class ofdiseases characterized by the uncontrolled growth of aberrant cells. Theterm includes all known cancers and neoplastic conditions, whethercharacterized as malignant, benign, soft tissue, or solid, and cancersof all stages and grades including pre- and post-metastatic cancers.Examples of different types of cancer include, but are not limited to,lung cancer (e.g., non-small cell lung cancer); digestive andgastrointestinal cancers such as colorectal cancer, gastrointestinalstromal tumors, gastrointestinal carcinoid tumors, colon cancer, rectalcancer, anal cancer, bile duct cancer, small intestine cancer, andstomach (gastric) cancer; esophageal cancer; gallbladder cancer; livercancer; pancreatic cancer; appendix cancer; breast cancer; ovariancancer; renal cancer (e.g., renal cell carcinoma); cancer of the centralnervous system; skin cancer; lymphomas; choriocarcinomas; head and neckcancers; osteogenic sarcomas; and blood cancers. As used herein, a“tumor” comprises one or more cancer cells or benign cells orprecancerous cells.

The term “decreased level of expression” as used herein, refers to adecrease in expression of a polynucleotide, e.g., gene, RNA, DNA, orprotein at least 10% or more. For example, 20%, 30%, 40%, or 50%, 60%,70%, 80%, 90% or more, or a decrease in expression of greater than1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold ormore as measured by one or more methods described herein. The term“increased level of expression” as used herein, refers to an increase inexpression of a polynucleotide, e.g., gene, RNA, DNA, or protein atleast 10% or more. For example, 20%, 30%, 40%, or 50%, 60%, 70%, 80%,90% or more or an increase in expression of greater than 1-fold, 2-fold,3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more as measuredby one or more methods, such as method described herein.

The term “diagnosis” refers to a process aimed at determining if anindividual is afflicted with a disease or ailment.

The term “hybridizing” refers to the binding of two single strandednucleic acids via complementary base pairing. The term “specifichybridization” refers to a process in which a nucleic acid moleculepreferentially binds, duplexes, or hybridizes to a particular nucleicacid sequence under stringent conditions (e.g., in the presence ofcompetitor nucleic acids with a lower degree of complementarity to thehybridizing strand). In certain embodiments of the present invention,these terms more specifically refer to a process in which a nucleic acidfragment (or segment) from a test sample preferentially binds to aparticular probe and to a lesser extent or not at all, to other probes,for example, when these probes are immobilized on an array.

The terms “labeled”, “labeled with a detectable agent” and “labeled witha detectable moiety” are used herein interchangeably. These terms areused to specify that an entity (e.g., a probe) can be visualized, forexample, following binding to another entity (e.g., a polynucleotide orpolypeptide). Preferably, the detectable agent or moiety is selectedsuch that it generates a signal which can be measured and whoseintensity is related to the amount of bound entity. In array-basedmethods, the detectable agent or moiety is also preferably selected suchthat it generates a localized signal, thereby allowing spatialresolution of the signal from each spot on the array. Methods forlabeling polypeptides or polynucleotides are well-known in the art.Labeled polypeptides or polynucleotides can be prepared by incorporationof or conjugation to a label, that is directly or indirectly detectableby spectroscopic, photochemical, biochemical, immunochemical,electrical, optical, or chemical means. Suitable detectable agentsinclude, but are not limited to, various ligands, radionuclides,fluorescent dyes, chemiluminescent agents, microparticles, enzymes,calorimetric labels, magnetic labels, and haptens. Detectable moietiescan also be biological molecules such as molecular beacons and aptamerbeacons.

The terms “normal” and “healthy” are used herein interchangeably. Theyrefer to an individual or group of individuals who have not shown tohave cancer or tumors. In certain embodiments, normal individuals havesimilar sex, age, body mass index as compared with the individual fromwhich the sample to be tested was obtained. The term “normal” is alsoused herein to qualify a sample isolated from a healthy individual.

The terms “nucleic acid molecule” and “polynucleotide” are used hereininterchangeably. They refer to a deoxyribonucleotide or ribonucleotidepolymer in either single- or double-stranded form, and unless otherwisestated, encompass known analogs of natural nucleotides that can functionin a similar manner as naturally occurring nucleotides. The termsencompass nucleic acid-like structures with synthetic backbones, as wellas amplification products.

As used herein, the term “gene” or “recombinant gene” refers to anucleic acid comprising an open reading frame encoding a polypeptide,including both exon and (optionally) intron sequences.

The term “genotype” as used herein includes to the genetic compositionof an organism, including, for example, whether a diploid organism isheterozygous or homozygous for one or more variant PIK3CA alleles ofinterest.

The term “probe”, as used herein, refers to a nucleic acid molecule ofknown sequence, which can be a short DNA sequence (i.e., anoligonucleotide), a PCR product, or mRNA isolate. Probes are specificDNA sequences to which nucleic acid fragments from a test sample arehybridized. Probes specifically bind to nucleic acids of complementaryor substantially complementary sequence through one or more types ofchemical bonds, usually through hydrogen bond formation.

The terms “protein”, “polypeptide”, and “peptide” are used hereininterchangeably, and refer to amino acid sequences of a variety oflengths, either in their neutral (uncharged) forms or as salts, andeither unmodified or modified by glycosylation, side chain oxidation, orphosphorylation. In certain embodiments, the amino acid sequence is thefull-length native protein. In other embodiments, the amino acidsequence is a smaller fragment of the full-length protein. In stillother embodiments, the amino acid sequence is modified by additionalsubstituents attached to the amino acid side chains, such as glycosylunits, lipids, or inorganic ions such as phosphates, as well asmodifications relating to chemical conversion of the chains, such asoxidation of sulfhydryl groups. Thus, the term “protein” (or itsequivalent terms) is intended to include the amino acid sequence of thefull-length native protein, subject to those modifications that do notchange its specific properties. In particular, the term “protein”encompasses protein isoforms, i.e., variants that are encoded by thesame gene, but that differ in their pI or MW, or both. Such isoforms candiffer in their amino acid sequence (e.g., as a result of alternativesplicing or limited proteolysis), or in the alternative, may arise fromdifferential post-translational modification (e.g., glycosylation,acylation, phosphorylation).

The term “protein analog”, as used herein, refers to a polypeptide thatpossesses a similar or identical function as the full-length nativeprotein but need not necessarily comprise an amino acid sequence that issimilar or identical to the amino acid sequence of the protein, orpossesses a structure that is similar or identical to that of theprotein. Preferably, in the context of the present invention, a proteinanalog has an amino acid sequence that is at least 30% (more preferably,at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% or at least 99%) identical to theamino acid sequence of the full-length native protein.

The term “protein fragment”, as used herein, refers to a polypeptidecomprising an amino acid sequence of at least 4 amino acid residues(preferably, at least 10 amino acid residues, at least 15 amino acidresidues, at least 20 amino acid residues, at least 25 amino acidresidues, at least 40 amino acid residues, at least 50 amino acidresidues, at least 60 amino acid residues, at least 70 amino acidresidues, at least 80 amino acid residues, at least 90 amino acidresidues, at least 100 amino acid residues, at least 125 amino acidresidues, at least 150 amino acid residues, at least 175 amino acidresidues, at least 200 amino acid residues, or at least 250 amino acidresidues) of the amino acid sequence of a second polypeptide. Thefragment of a marker protein may or may not possess a functionalactivity of the full-length native protein.

The term “subject,” “individual,” and “patient” are used interchangeablyherein to mean a human or other animal, such as farm animals orlaboratory animals (e.g., guinea pig or mice) capable of having cellcycle (influenced) determined diseases, either naturally occurring orinduced, including but not limited to cancer.

The term “sensitize” as used herein means to alter cancer cells or tumorcells in a way that allows for more effective treatment of theassociated neoplastic disease with an antimetabolite agent, ananticancer agent, or radiation therapy. In some embodiments, normalcells are not affected to an extent that causes the normal cells to beunduly injured by the antimetabolite, chemotherapy, or radiationtherapy.

A “single nucleotide polymorphism” or “SNP” occurs at a polymorphic siteoccupied by a single nucleotide, which is the site of variation betweenallelic sequences. The site is usually preceded by and followed byhighly conserved sequences of the allele (e.g., sequences that vary inless than 1/100 or 1/1000 members of the populations). A SNP usuallyarises due to substitution of one nucleotide for another at thepolymorphic site, and occurs in at least 1% of the population.

The term “synergistic effect” as used herein means the combined effectof two or more anticancer agents or chemotherapy drugs can be greaterthan the sum of the separate effects of the anticancer agents orchemotherapy drugs alone.

A “therapeutically effective amount” of a therapeutic agent is an amountthat achieves the intended therapeutic effect of reducing cancerouscells, precancerous cells or benign tumor cells having a PIK3CA proteinor gene mutation in a subject. The full therapeutic effect does notnecessarily occur by administration of one dose and may occur only afteradministration of a series of doses. Thus, a therapeutically effectiveamount may be administered in one or more administrations.

A “prophylactically effective amount” of a therapeutic agent is anamount of a therapeutic agent that, when administered to a subject, willhave the intended prophylactic effect, e.g., preventing or delaying theonset (or reoccurrence) of the disease or symptoms, or reducing thelikelihood of the onset (or reoccurrence) of the disease or symptoms.The full prophylactic effect does not necessarily occur byadministration of one dose and may occur only after administration of aseries of doses. Thus, a prophylactically effective amount may beadministered in one or more administrations.

An “effective amount” of a therapeutic agent is an amount that producesthe desired effect.

“Treating” cancer in a patient refers to taking steps to obtainbeneficial or desired results, including clinical results. For purposesof this invention, beneficial or desired clinical results include, butare not limited to alleviation or amelioration of one or more symptomsof the cancer; diminishing the extent of disease; delaying or slowingdisease progression; amelioration and palliation or stabilization of thedisease state.

The term “wild type” (wt) cell or cell line is used herein, for purposesof the specification and claims, to mean a cell or cell line thatretains the characteristics normally associated with that type of cellor cell line for the physiological process or morphologicalcharacteristic that is being examined. It is permissible for the cell orcell line to have non-wild type characteristics for physiologicalprocess or morphological characteristics that are not being examined aslong as they do not appreciably affect the process or characteristicbeing examined.

The term “mutant” refers to any change in the genetic material of anorganism, in particular a change (i.e., deletion, substitution,addition, or alteration) in a wild type polynucleotide sequence or anychange in a wild type protein. The term “variant” is usedinterchangeably with “mutant”. Although it is often assumed that achange in the genetic material results in a change of the function ofthe protein, the terms “mutant” and “variant” refer to a change in thesequence of a wild type protein regardless of whether that change altersthe function of the protein (e.g., increases, decreases, imparts a newfunction), or whether that change has no effect on the function of theprotein (e.g., the mutation or variation is silent).

Embodiments described herein relate to methods of determining thesusceptibility, resistance, responsiveness, and/or sensitivity of acancer, precancerous cells or a benign tumor in a subject to treatmentwith an inhibitor of one more enzymes of the glutamine metabolismpathway by determining the presence of a mutated PIK3CA gene or a mutantform of PIK3CA protein or a biologically active fragment thereof and/orthe level of GPT2 expression in a sample of cancer cells, precancerouscells or benign tumor cells obtained from the subject. It was found thatPIK3CA mutations reprogram glutamine metabolism in cancer cells byup-regulating glutamate pyruvate transaminase 2 (GPT2), therebyrendering them more dependent on glutamine. Compared to isogenicwild-type (WT) cells, PIK3CA mutant cancer cells convert substantiallymore glutamine to α-ketoglutarate in order to replenish thetricarboxylic acid (TCA) cycle and generate ATP. Mutant p110αup-regulates GPT2 gene expression through an AKT-independentPDK1-RSK2-ATF4 signaling axis. Moreover, inhibitors that target one ormore glutamine metabolism enzymes, such as glutaminases or glutamatemetabolism enzymes, including GPT2, can suppress tumor growth of cancercells with PIK3CA mutations, but not cancer cells with WT PIK3CA.

Advantageously, the identification of cancer cells harboring PIK3CAmutations can be used as a predictive marker to determine thesusceptibility, resistance, responsiveness, and/or sensitivity of thecancer cells to treatment with an inhibitor of one more enzymes of theglutamine metabolism pathway. Targeting one more enzymes of theglutamine metabolism pathway can thereby afford new therapies for thetreatment of patients whose cancers harbor PIK3CA mutations.

In some embodiments, a method of determining susceptibility, resistance,responsiveness, and/or sensitivity to a cancer, precancerous cells,and/or benign tumor is a subject thereof to inhibitors of one or moreenzymes of the glutamine metabolism pathway can include obtaining asample of the cancer cells, the precancerous cells or the benign tumorcells from the subject, assaying the cells in the sample for thepresence of a mutated PIK3CA gene or a mutant form of PIK3CA protein ora biologically active fragment thereof, and determining that the subjectshould be treated with the inhibitor if the cancer cells have themutated PIK3CA gene or the mutant form of PIK3CA protein. In otherembodiments, the method can include obtaining a sample of the cancercells, the precancerous cells or the benign tumor cells from thesubject, measuring the level of GPT2 expression in the cancer cells,comparing the measured level of GPT2 expression in the cancer cells to acontrol level, and identifying the cancer is more susceptible totreatment with the inhibitor if there is an increase in the measuredlevels of GPT2 expression in the cancer cells compared to a controllevel.

In some embodiments, the presence of mutated PIK3CA gene or a mutantform of PIK3CA protein and/or the level of GPT2 expression in the cancercells of the subject can be determined by obtaining a sample of cancercells from the subject diagnosed with cancer and determining thepresence of mutated PIK3CA gene or a mutant form of PIK3CA proteinand/or the level of GPT2 expression in the cancer cells. Cancer (andprecancerous lesions) can include any tumor or cancerous cell that has aPIK3CA mutation. Such cancers include breast cancer, neuroblastoma,gastrointestinal carcinoma such as rectum carcinoma, colon carcinoma,familial adenomatous polyposis carcinoma and hereditary non-polyposiscolorectal cancer, esophageal carcinoma, labial carcinoma, larygialcarcinoma, hypopharyngial carcinoma, tongue carcinoma, salivary glandcarcinoma, gastric carcinoma, medullary thyroid carcinoma, papillarythyroid carcinoma, renal carcinoma, kidney parenchymal carcinoma,ovarian carcinoma, cervical carcinoma, uterine corpus carcinoma,endometrium carcinoma, choriocarcinoma, pancreatic carcinoma, prostatecarcinoma, testis carcinoma, urinary carcinoma, melanoma, brain tumorssuch as glioblastoma, astrocytoma, meningioma, medulloblastoma andperipheral neuroectodermal tumors, Hodgkin's lymphoma, non-Hodgkin'slymphoma, Burkitt's lymphoma, acute lymphocytic leukemia (ALL), chroniclymphocytic leukemia (CLL), acute myelologenous leukemia (AML), chronicmyelologenous leukemia (CML), adult T-cell leukemia/lymphoma,hepatocellular carcinoma, gallbladder carcinoma, bronchial carcinoma,small cell lung carcinoma, non-small cell lung carcinoma, multiplemyeloma, basal cell carcinoma, teratoma, retinoblastoma, choroidalmelanoma, seminoma, rhabdomyosarcoma, craniopharyngioma, osteosarcoma,chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing's sarcomaand plasmocytoma. Particular tumors include those of the brain, liver,kidney, bladder, breast, gastric, ovarian, colorectal, prostate,pancreatic, lung, vulval, thyroid, colorectal, oesophageal, sarcomas,glioblastomas, head and neck, leukemias and lymphoid malignancies. Insome embodiments, the cancer can be selected from the group consistingof carcinomas, melanomas, sarcomas, lymphomas, leukemias, astrocytomas,gliomas, malignant melanomas, chronic lymphocytic leukemia, lungcancers, prostate cancer, colorectal cancers, ovarian cancers,pancreatic cancers, renal cancers, endometrial cancers, gastric cancers,liver cancers, head and neck cancers.

The samples used in the practice of the inventive methods may be freshor frozen samples collected from a subject, or archival samples.Biological samples may be collected by any non-invasive means, such as,for example, by drawing blood from a subject, or using fine needleaspiration or needle biopsy. Alternatively, biological samples may becollected by an invasive method, including, for example, surgicalbiopsy.

In certain embodiments, the inventive methods are performed on thebiological sample itself without or with limited processing of thesample.

In some embodiments, mutated PIK3CA genes or gene products and/or GPT2expression levels can be detected in tumor samples or, in some types ofcancer, in biological samples such as urine, stool, sputum or serum. Forexample, serum has been tested in the context of colorectal cancer.Cancer cells are found in blood and serum for cancers, such as lymphomaor leukemia. The same techniques discussed above for detection of mutantPIK3CA genes or gene products in tumor samples can be applied to otherbody samples. Cancer cells are sloughed off from tumors and appear insuch body samples.

In other embodiments, the inventive methods are performed at the singlecell level (e.g., isolation of cells from a biological sample). However,in such embodiments, the inventive methods are preferably performedusing a sample comprising many cells, where the assay is “averaging”expression over the entire collection of cells present in the sample.Preferably, there is enough of the biological sample to accurately andreliably determine mutated PIK3CA genes or gene products and/or GPT2expression levels. Multiple biological samples may be taken from thesame tissue/body part in order to obtain a representative sampling ofthe tissue.

In still other embodiments, the mutated PIK3CA genes or gene productsand/or GPT2 expression levels can be measured in a protein extractprepared from cancer cells of a biological sample. The protein extractcan contain the total PIK3CA and/or GPT2 content by the cancer cell orcells. Methods of protein extraction are well known in the art (see, forexample “Protein Methods”, D. M. Bollag et al., 2nd Ed., 1996,Wiley-Liss; “Protein Purification Methods: A Practical Approach”, E. L.Harris and S. Angal (Eds.), 1989; “Protein Purification Techniques: APractical Approach”, S. Roe, 2nd Ed., 2001, Oxford University Press;“Principles and Reactions o/Protein Extraction, Purification, andCharacterization”, H. Ahmed, 2005, CRC Press: Boca Raton, Fla.).Numerous different and versatile kits can be used to extract proteinsfrom cells, and are commercially available from, for example, BioRadLaboratories (Hercules, Calif.), BD Biosciences Clontech (Mountain View,Calif.), Chemicon International, Inc. (Temecula, Calif.), Calbiochem(San Diego, Calif.), Pierce Biotechnology (Rockford, Ill.), andInvitrogen Corp. (Carlsbad, Calif.). User Guides that describe in greatdetail the protocol to be followed are usually included in all thesekits. Sensitivity, processing time and costs may be different from onekit to another. One of ordinary skill in the art can easily select thekits) most appropriate for a particular situation. After the proteinextract has been obtained, the protein concentration of the extract canbe standardized to a value being the same as that of the control samplein order to allow signals of the PIK3CA and/or GPT2 expression levels tobe quantitated. Such standardization can be made using photometric orspectrometric methods or gel electrophoresis.

In yet other embodiments, mutated PIK3CA genes or gene products and/orGPT2 expression levels can be measured from nucleic acid moleculesextracted from cancer cells of a biological sample. For example, RNA maybe extracted from the sample before analysis. Methods of RNA extractionare well known in the art (see, for example, J. Sambrook et al.,“Molecular Cloning: A Laboratory Manual”, 1989, 2nd Ed., Cold SpringHarbor Laboratory Press: Cold Spring Harbor, N.Y.). Most methods of RNAisolation from cells are based on the disruption of the tissue in thepresence of protein denaturants to quickly and effectively inactivateRNAses. Isolated total RNA may then be further purified from the proteincontaminants and concentrated by selective ethanol precipitations,phenol/chloroform extractions followed by isopropanol precipitation orcesium chloride, lithium chloride or cesium trifluoroacetate gradientcentrifugations. Kits are also available to extract RNA (i.e., total RNAor mRNA) from bodily fluids or tissues and are commercially availablefrom, for example, Ambion, Inc. (Austin, Tex.), Amersham Biosciences(Piscataway, N.J.), BD Biosciences Clontech (Palo Alto, Calif.), BioRadLaboratories (Hercules, Calif.), GIBCO BRL (Gaithersburg, Md.), andQiagen, Inc. (Valencia, Calif.).

In certain embodiments, after extraction, mRNA is amplified, andtranscribed into cDNA, which can then serve as template for multiplerounds of transcription by the appropriate RNA polymerase. Amplificationmethods are well known in the art (see, for example, A. R. Kimmel and S.L. Berger, Methods Enzymol. 1987, 152: 307-316; J. Sambrook et al.,“Molecular Cloning: A Laboratory Manual”, 1989, 2nd Ed., Cold SpringHarbour Laboratory Press: New York; “Short Protocols in MolecularBiology”, F. M. Ausubel (Ed.), 2002, 5th Ed., John Wiley & Sons; U.S.Pat. Nos. 4,683,195; 4,683,202 and 4,800,159). Reverse transcriptionreactions may be carried out using non-specific primers, such as ananchored oligo-dT primer, or random sequence primers, or using atarget-specific primer complementary to the RNA, or using thermostableDNApolymerases (such as avian myeloblastosis virus reverse transcriptaseor Moloney murine leukemia virus reverse transcriptase).

In general, mutated PIK3CA genes or gene products and/or GPT2 expressionlevels in the cancer cells can be determined by contacting cancer cellsin a biological sample isolated from a subject with binding agents forPIK3CA and/or GPT2; detecting, in the sample, the presence or levels ofthe mutated PIK3CA genes or gene products and/or GPT2 that bind to thebinding agents; and optionally, comparing the detected mutated PIK3CAgenes or gene products and/or GPT2 expression levels in the sample withthe levels of mutated PIK3CA genes or gene products and/or GPT2 in acontrol sample. As used herein, the term “binding agent” refers to anentity, such as a polypeptide or antibody that specifically binds tomutated PIK3CA genes or gene products and/or GPT2. An entity“specifically binds” to mutated PIK3CA genes or gene products and/orGPT2 if it reacts/interacts at a detectable level with mutated PIK3CAgenes or gene products and/or GPT2 but does not react/interactdetectably with polynucleotides and/or peptides containing unrelatedsequences or sequences of different polypeptides.

In certain embodiments, the binding agent is an RNA molecule, or apolypeptide (e.g., a polypeptide that comprises a polypeptide sequenceof a protein marker, a peptide variant thereof, or a non-peptide mimeticof such a sequence).

In other embodiments, the binding agent is an antibody specific formutated PIK3CA and/or GPT2. Antibodies for use in the methods includemonoclonal and polyclonal antibodies, immunologically active fragments(e.g., Fab or (Fab)2 fragments), antibody heavy chains, humanizedantibodies, antibody light chains, and chimeric antibodies. Antibodies,including monoclonal and polyclonal antibodies, fragments and chimeras,may be prepared using methods known in the art (see, for example, R. G.Mage and E. Lamoyi, in “Monoclonal Antibody Production Techniques andApplications”, 1987, Marcel Dekker, Inc.: New York, pp. 79-97; G. Kohlerand C. Milstein, Nature, 1975, 256: 495-497; D. Kozbor et al., J.Immunol. Methods, 1985, 81: 31-42; and R. J. Cote et al., Proc. Natl.Acad. Sci. 1983, 80: 2026-203; R. A. Lerner, Nature, 1982, 299: 593-596;A. C. Nairn et al., Nature, 1982, 299: 734-736; A. J. Czernik et al.,Methods Enzymol. 1991, 201: 264-283; A. J. Czernik et al., Neuromethods:Regulatory Protein Modification: Techniques & Protocols, 1997, 30:219-250; A. J. Czemik et al., NeuroNeuroprotocols, 1995, 6: 56-61; H.Zhang et al., J. Biol. Chem. 2002, 277: 39379-39387; S. L. Morrison etal., Proc. Natl. Acad. Sci., 1984, 81: 6851-6855; M. S. Neuberger etal., Nature, 1984, 312: 604-608; S. Takeda et al., Nature, 1985, 314:452-454). Antibodies to be used in the methods can be purified bymethods well known in the art (see, for example, S. A. Minden,“Monoclonal Antibody Purification”, 1996, IBC Biomedical Library Series:Southbridge, Mass.). For example, antibodies can be affinity purified bypassage over a column to which a protein marker or fragment thereof isbound. The bound antibodies can then be eluted from the column using abuffer with a high salt concentration.

Instead of being prepared, antibodies to be used in the methodsdescribed herein may be obtained from scientific or commercial sources.

In certain embodiments, the binding agent is directly or indirectlylabeled with a detectable moiety. The role of a detectable agent is tofacilitate the measuring of the mutated PIK3CA genes or gene productsand/or GPT2 expression levels by allowing visualization of the complexformed by binding of the binding agent to mutated PIK3CA genes or geneproducts and/or GPT2 expression levels (or analog or fragment thereof).The detectable agent can be selected such that it generates a signalwhich can be measured and whose intensity is related (preferablyproportional) to the presence and/or amount of mutated PIK3CA genes orgene products and/or GPT2 expression levels present in the sample beinganalyzed. Methods for labeling biological molecules such as polypeptidesand antibodies are well-known in the art (see, for example, “AffinityTechniques. Enzyme Purification. Part B”, Methods in Enzymol., 1974,Vol. 34, W. B. Jakoby and M. Wilneck (Eds.), Academic Press: New York,N.Y.; and M. Wilchek and E. A. Bayer, Anal. Biochem., 1988, 171: 1-32).

Any of a wide variety of detectable agents can be used in the methodsdescribed herein. Detectable agents include, but are not limited to:various ligands, radionuclides, fluorescent dyes, chemiluminescentagents, microparticles (such as, for example, quantum dots,nanocrystals, phosphors and the like), enzymes (such as, for example,those used in an ELISA, i.e., horseradish peroxidase,beta-galactosidase, luciferase, alkaline phosphatase), colorimetriclabels, magnetic labels, and biotin, dioxigenin or other haptens andproteins for which antisera or monoclonal antibodies are available.

In certain embodiments, the binding agents (e.g., antibodies) may beimmobilized on a carrier or support (e.g., a bead, a magnetic particle,a latex particle, a microtiter plate well, a cuvette, or other reactionvessel). Examples of suitable carrier or support materials includeagarose, cellulose, nitrocellulose, dextran, Sephadex, Sepharose,liposomes, carboxymethyl cellulose, polyacrylamides, polystyrene,gabbros, filter paper, magnetite, ion-exchange resin, plastic film,plastic tube, glass, polyamine-methyl vinylether-maleic acid copolymer,amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, andthe like. Binding agents may be indirectly immobilized using secondbinding agents specific for the first binding agents (e.g., mouseantibodies specific for the protein markers may be immobilized usingsheep anti-mouse IgG Fc fragment specific antibody coated on the carrieror support).

Mutated PIK3CA and/or GPT2 expression levels in the methods describedherein may be determined using immunoassays. Examples of such assays areradioimmunoassays, enzyme immunoassays (e.g., ELISA), immunofluorescenceimmunoprecipitation, latex agglutination, hemagglutination, andhistochemical tests, which are conventional methods well-known in theart. As will be appreciated by one skilled in the art, the immunoassaymay be competitive or noncompetitive. Methods of detection andquantification of the signal generated by the complex formed by bindingof the binding agent with the mutated PIK3CA and/or GPT2 will depend onthe nature of the assay and of the detectable moiety (e.g., fluorescentmoiety).

Alternatively, mutated PIK3CA and/or GPT2 expression levels may bedetermined using mass spectrometry based methods or image (including useof labeled ligand) based methods known in the art for the detection ofproteins. Other suitable methods include proteomics-based methods.Proteomics, which studies the global changes of protein expression in asample, typically includes the following steps: (I) separation ofindividual proteins in a sample by electrophoresis (2-D PAGE), (2)identification of individual proteins recovered from the gel (e.g., bymass spectrometry or N-terminal sequencing), and (3) analysis of thedata using bioinformatics.

As already mentioned above, the methods described herein may involvedetermination of the expression levels of a set of nucleic acidmolecules comprising polynucleotide sequences coding for mutated PIK3CAgenes or gene products and/or GPT2. Determination of the presence and/orexpression levels of nucleic acid molecules in the practice of theinventive methods may be performed by any method, including, but notlimited to, Southern analysis, Northern analysis, polymerase chainreaction (PCR) (see, for example, U.S. Pat. Nos. 4,683,195; 4,683,202,and 6,040,166; “PCR Protocols: A Guide to Methods and Applications”,Innis et al. (Eds.), 1990, Academic Press: New York), reversetranscriptase PCR(RT-PCT), anchored PCR, competitive PCR (see, forexample, U.S. Pat. No. 5,747,251), rapid amplification of cDNA ends(RACE) (see, for example, “Gene Cloning and Analysis: CurrentInnovations, 1997, pp. 99-115); ligase chain reaction (LCR) (see, forexample, EP 01 320308), one-sided PCR (Ohara et al., Proc. Natl. Acad.Sci., 1989, 86: 5673-5677), in situ hybridization, Taqman based assays(Holland et al., Proc. Natl. Acad. Sci., 1991, 88:7276-7280),differential display (see, for example, Liang et al., Nucl. Acid. Res.,1993, 21: 3269-3275) and other RNA fingerprinting techniques, nucleicacid sequence based amplification (NASBA) and other transcription basedamplification systems (see, for example, U.S. Pat. Nos. 5,409,818 and5,554,527), Qbeta Replicase, Strand Displacement Amplification (SDA),Repair Chain Reaction (RCR), nuclease protection assays,subtraction-based methods, Rapid-Scan™, and the like.

Nucleic acid probes for use in the detection of polynucleotide sequencesin biological samples may be constructed using conventional methodsknown in the art. Probes may be based on nucleic acid sequences encodingat least 5 sequential amino acids from regions of nucleic acids encodingmutated PIK3CA genes or gene products and/or GPT2, and preferablycomprise about 15 to about 50 nucleotides. A nucleic acid probe may belabeled with a detectable moiety, as mentioned above in the case ofbinding agents. The association between the nucleic acid probe anddetectable moiety can be covalent or non-covalent. Detectable moietiescan be attached directly to nucleic acid probes or indirectly through alinker (E. S. Mansfield et al., Mol. Cell. Probes, 1995, 9: 145-156).Methods for labeling nucleic acid molecules are well-known in the art(for a review of labeling protocols, label detection techniques andrecent developments in the field, see, for example, L. J. Kricka, AnnClin. Biochem. 2002, 39: 114-129; R. P. van Gijlswijk et al., ExpertRev. Mol. Diagn. 2001, 1: 81-91; and S. Joos et al., J. Biotechnol.1994, 35:135-153).

Nucleic acid probes may be used in hybridization techniques to detectpolynucleotides encoding mutated PIK3CA genes or gene products and/orGPT2. The technique generally involves contacting an incubating nucleicacid molecules in a biological sample obtained from a subject with thenucleic acid probes under conditions such that specific hybridizationtakes place between the nucleic acid probes and the complementarysequences in the nucleic acid molecules. After incubation, thenon-hybridized nucleic acids are removed, and the presence and amount ofnucleic acids that have hybridized to the probes are detected andquantified.

Detection of nucleic acid molecules comprising polynucleotide sequencescoding for mutated PIK3CA genes or gene products and/or GPT2 may involveamplification of specific polynucleotide sequences using anamplification method such as PCR, followed by analysis of the amplifiedmolecules using techniques known in the art. Suitable primers can beroutinely designed by one skilled in the art. In order to maximizehybridization under assay conditions, primers and probes employed in themethods of the invention generally have at least 60%, preferably atleast 75% and more preferably at least 90% identity to a portion ofnucleic acids encoding a protein marker.

Primer sequences and amplification protocols for evaluating PIK3CAmutations are known to those in the art and have been published.Examples include Karakas, et al., Mutation of the PIK3CA Oncogene inHuman Cancers, BRITISH J CANCER 94(4):455-459 (2006); Li et al.,Mutations of PIK3CA in Gastric Adenocarcinoma, BIOMED CENTRAL CANCER5:29 (2005); Qiu et al., PIK3CA Mutations in Head and Neck Squamous CellCarcinoma, CLIN CANCER RES. 12(5):1441-1446 (2006). The most frequentPIK3CA mutations are E542K (Glu524Lys), E545K (Glu545Lys), and E545D(Glu545Asp) mutations in exon 9 and H1047R (His1047Arg) mutations inexon 20.

Hybridization and amplification techniques described herein may be usedto assay qualitative and quantitative aspects of expression of nucleicacid molecules comprising polynucleotide sequences coding for theinventive gene or protein markers.

Alternatively, oligonucleotides or longer fragments derived from nucleicacids encoding each protein marker may be used as targets in amicroarray. A number of different array configurations and methods oftheir production are known to those skilled in the art (see, forexample, U.S. Pat. Nos. 5,445,934; 5,532,128; 5,556,752; 5,242,974;5,384, 261; 5,405,783; 5,412,087; 5,424,186; 5,429,807; 5,436,327;5,472,672; 5,527,681; 5,529,756; 5,545,531; 5,554, 501; 5,561,071;5,571,639; 5,593,839; 5,599,695; 5,624,711; 5,658,734; and 5,700,637).Microarray technology allows for the measurement of the steady-statelevel of large numbers of polynucleotide sequences simultaneously.Microarrays currently in wide use include cDNA arrays andoligonucleotide arrays. Analyses using microarrays are generally basedon measurements of the intensity of the signal received from a labeledprobe used to detect a cDNA sequence from the sample that hybridizes toa nucleic acid probe immobilized at a known location on the microarray(see, for example, U.S. Pat. Nos. 6,004,755; 6,218,114; 6,218,122; and6,271,002). Array-based gene expression methods are known in the art andhave been described in numerous scientific publications as well as inpatents (see, for example, M. Schena et al., Science, 1995, 270:467-470; M. Schena et al., Proc. Natl. Acad. Sci. USA 1996, 93:10614-10619; 1.1. Chen et al., Genomics, 1998, 51: 313324; U.S. Pat.Nos. 5,143,854; 5,445,934; 5,807,522; 5,837, 832; 6,040,138; 6,045,996;6,284,460; and 6,607,885).

In some embodiments, a mutation in the PIK3CA gene in a sample can bedetected by amplifying nucleic acid corresponding to the PIK3CA geneobtained from the sample, or a biologically active fragment, andcomparing the electrophoretic mobility of the amplified nucleic acid tothe electrophoretic mobility of corresponding wild-type PIK3CA gene orfragment thereof. A difference in the mobility indicates the presence ofa mutation in the amplified nucleic acid sequence. Electrophoreticmobility may be determined on polyacrylamide gel. Alternatively, anamplified PIK3CA gene or fragment nucleic acid may be analyzed fordetection of mutations using Enzymatic Mutation Detection (EMD) (DelTito et al, Clinical Chemistry 44:731-739, 1998). EMD uses thebacteriophage resolvase T₄ endonuclease VII, which scans alongdouble-stranded DNA until it detects and cleaves structural distortionscaused by base pair mismatches resulting from point mutations,insertions and deletions. Detection of two short fragments formed byresolvase cleavage, for example by gel eletrophoresis, indicates thepresence of a mutation. Benefits of the EMD method are a single protocolto identify point mutations, deletions, and insertions assayed directlyfrom PCR reactions eliminating the need for sample purification,shortening the hybridization time, and increasing the signal-to-noiseratio. Mixed samples containing up to a 20-fold excess of normal DNA andfragments up to 4 kb in size can been assayed.

In other embodiments, the ligase chain reaction, which is known in theart, can also be used to amplify PIK3CA sequences. In addition, atechnique known as allele specific PCR can be used. According to thistechnique, primers are used which hybridize at their 3′ ends to aparticular PIK3CA mutation. If the particular PIK3CA mutation is notpresent, an amplification product is not observed. AmplificationRefractory Mutation System (ARMS) can also be used as disclosed inEuropean Patent Application Publication No. 0332435 and in Newton etal., Nucleic Acids Research, Vol. 17, p. 7, 1989. Insertions anddeletions of genes can also be detected by cloning, sequencing andamplification. In addition, restriction fragment length polymorphism,(RFLP) probes for the gene or surrounding marker genes can be used toscore alteration of an allele or an insertion in a polymorphic fragment.Single stranded conformation polymorphism (SSCP) analysis can also beused to detect base change variants of an allele. (Orita et al., Proc.Natl. Acad. Sci. USA Vol. 86, pp. 2766-2770, 1989, and Genomics, Vol. 5,pp. 874-879, 1989). Other techniques for detecting insertions anddeletions as are known in the art can be used.

Mismatches can include hybridized nucleic acid duplexes which are not100% complementary. The lack of total complementarity may be due todeletions, insertions, inversions, substitutions or frameshiftmutations. Mismatch detection can be used to detect point mutations inthe gene or its mRNA product. While these techniques are less sensitivethan sequencing, they are simpler to perform on a large number of tumorsamples. An example of a mismatch cleavage technique is the RNaseprotection method, which is described in detail in Winter et al., Proc.Natl. Acad. Sci. USA, Vol. 82, p. 7575, 1985 and Meyers et al., Science,Vol. 230, p. 1242, 1985. A labeled riboprobe which is complementary tothe human wild-type PIK3CA gene coding sequence can also be used. Theriboprobe and either mRNA or DNA isolated from the tumor tissue areannealed (hybridized) together and subsequently digested with the enzymeRNase A which is able to detect some mismatches in a duplex RNAstructure. If a mismatch is detected by RNase A, it cleaves at the siteof the mismatch. Thus, when the annealed RNA preparation is separated onan electrophoretic gel matrix, if a mismatch has been detected andcleaved by RNase A, an RNA product will be seen which is smaller thanthe full-length duplex RNA for the riboprobe and the mRNA or DNA. Theriboprobe need not be the full length of the PIK3CA mRNA or gene. If theriboprobe comprises only a segment of the PIK3CA mRNA or gene it will bedesirable to use a number of these probes to screen the whole mRNAsequence for mismatches.

In a similar manner, DNA probes can be used to detect mismatches,through enzymatic or chemical cleavage. See, e.g., Cotton et al., Proc.Natl. Acad. Sci. USA, Vol. 85, 4397, 1988; and Shenk et al., Proc. Natl.Acad. Sci. USA, Vol. 72, p. 989, 1975. Alternatively, mismatches can bedetected by shifts in the electrophoretic mobility of mismatchedduplexes relative to matched duplexes. See, e.g., Cariello, HumanGenetics, Vol. 42, p. 726, 1988. With either riboprobes or DNA probes,the cellular mRNA or DNA which might contain a mutation can be amplifiedusing PCR before hybridization. Changes in DNA of the PIK3CA gene canalso be detected using Southern hybridization, especially if the changesare gross rearrangements, such as deletions and insertions.

Once the mutated PIK3CA genes or gene products and/or GPT2 expressionlevels in the cancer cells has been measured or determined (as describedabove), the measured mutated PIK3CA genes or gene products and/or GPT2expression levels can optionally be compared to a control level. Thecontrol level can be based upon the level of mutated PIK3CA and/or GPT2in a normal cell obtained from a control population (e.g., the generalpopulation) or a select population of subjects. For example, the selectpopulation may be comprised of apparently healthy subjects or fromsubjects at risk of developing cancer.

The control level can be related to the value used to characterize thelevel of mutated PIK3CA and/or GPT2 expression levels obtained from thesubject. The control level can also take a variety of forms. Forexample, the control level can be a single cut-off value, such as amedian or mean. The control level can be established based uponcomparative groups, such as where the level in one defined group isdouble the level of another defined group.

Control levels of mutated PIK3CA and/or GPT2 expression in cells, forexample, can be obtained (e.g., mean levels, median levels, or “cut-off”levels) by assaying a large sample of subjects in the general populationor a select population and then using a statistical model, such as thepredictive value method for selecting a positivity criterion or receiveroperator characteristic curve that defines optimum specificity (highesttrue negative rate) and sensitivity (highest true positive rate), asdescribed in Knapp, R. G. and Miller, M. C. (1992): ClinicalEpidemiology and Biostatistics, William and Wilkins, Harual PublishingCo. (Malvern, Pa.).

Depending upon the level or value of measured mutated PIK3CA and/or GPT2when compared to the control level, a determination can be made as towhether the cancer cells or cancer of the subject is more or lesssusceptible, sensitive, and/or resistance to treatment with an inhibitorof one or more enzymes of the glutamine metabolism pathway. In someembodiments, a determined presence of a mutated PIK3CA gene or a mutantform of PIK3CA protein or a biologically active fragment thereof for thecancer identifies the cancer as being more susceptible to treatment withthe inhibitor of one or more enzymes of the glutamine metabolismpathway. An absence of a mutated PIK3CA gene or a mutant form of PIK3CAprotein or a biologically active fragment thereof for the canceridentifies the cancer as being less susceptible to treatment with theinhibitor of one or more enzymes of the glutamine metabolism pathway. Inother embodiments, a GPT2 expression level higher or increased comparedto the control level identifies the cancer as being more susceptible totreatment with the inhibitor of one or more enzymes of the glutaminemetabolism pathway. In contrast, a measured or determined expressionlevel of GPT2 expression less than the control level identifies thecancer as being less susceptible to treatment with the inhibitor of oneor more enzymes of the glutamine metabolism pathway.

By determining the efficacy of the inhibitor of one or more enzymes ofthe glutamine metabolism pathway, such as inhibitors of glutaminaseand/or inhibitors of aminotransferase (e.g., glutamate pyruvatetransaminase, aspirate aminotransferase, and glutamate dehydrogenase),to treating cancer and/or susceptibility, sensitivity, responsiveness,and/or resistance of the cancer cell to the inhibitor, skilledphysicians may select and prescribe treatments adapted to eachindividual patient with increased efficiency. In some embodiments, amethod of treating cancer with the inhibitors described herein, such asglutaminase inhibitors and/or aminotransferase inhibitors, can includefirst determining the presence of mutated PIK3CA genes or gene productsand/or GPT2 expression levels of cancer cells of a subject diagnosedwith cancer and then administering an inhibitor of one more enzymes ofthe glutamine metabolism pathway, depending on the determined ormeasured presence of mutated PIK3CA genes or gene products and/or GPT2expression levels.

In some embodiments, an inhibitor of one or more enzymes of theglutamine metabolism pathway can be a glutaminase inhibitor and/oraminotransferase inhibitor. Examples of glutaminase inhibitors caninclude heterocyclic inhibitors of glutaminase having formula Idisclosed in U.S. Pat. No. 8,604,016, and U.S. Patent ApplicationPublication Nos. 2014/0050699A1 and 2015/0004134, which are hereinincorporated by reference in their entirety. Heterocyclic inhibitors ofglutaminase having formula I disclosed in U.S. Pat. No. 8,604,016 caninclude of the compounds disclosed in Table 3 of the application. Insome embodiments, the glutaminase inhibitor can include CB-839 or apharmaceutically acceptable salt thereof. In other embodiments, theglutaminase inhibitor has the formula:

or a pharmaceutically acceptable salt thereof.

Other examples of glutaminase inhibitors includebis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) andanalogs thereof;N-(5-{2-[2-(5-amino-[1,2,4]thiadiazol-2-yl)-ethylsulfanyl]-ethyl}-[1,3,4]-thiadiazol-2-yl)-2-phenyl-acetamide;small molecule 968 and derivatives thereof 6-diazo-5-oxo-L-norleucine(DON); N-ethylmaleimide (NEM); p-chloromercuriphenylsulfonate (pCMPS);L-2-amino-4-oxo-5-chloropentoic acid; DON plus o-carbamoyl-L-serine;acivicin [(alphaS,5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleaceticacid]; azaserine; and5-β-bromo-4-(dimethylamino)phenyl)-2,2-dimethyl-2,3,5,6-tetrahydrobenzo[-a]phenanthridin-4(1H)-oneStill other examples of glutaminase inhibitors include imidazolederivatives having formula I disclosed in U.S. Pat. No. 5,552,427, whichis herein incorporated by reference in its entirety.

In some embodiments, the aminotransferase inhibitor can be a selectiveor partially glutamate pyruvate transanimase (GPT) or an alanineaminotransferase inhibitor. An example of alanine aminotransferaseinhibitor is aminooxyacetate Aminooxyacetate inhibits enzymatic activityof amino transaminases including GPT2. Other examples ofaminotransferase inhibitors, such as GPT2 inhibitors, includeL-cycloserine and β-chloro-L-alanine, and epigallocatechin gallate(EGCG).

In some embodiments, the glutaminase inhibitor and/or theaminotransferase inhibitor can be administered to subject having cancer,precancerous cells or a benign tumor with mutated PIK3CA genes or geneproducts and/or elevated GPT2 expression levels at therapeuticallyeffective amounts. The therapeutically effective can be an amounteffective to substantially inhibit glutamine metabolism in the cancercells and/or suppress cancer cell growth and/or proliferation.

The inhibitors of one or more enzymes of the glutamine metabolismpathway, including glutaminase inhibitors and/or the aminotransferaseinhibitors, can be administered in a number of ways depending uponwhether local or systemic treatment is desired and upon the area to betreated. Administration may be topical (including ophthalmic and tomucous membranes including vaginal and rectal delivery), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. In some embodiments a slow release preparationcomprising the therapeutic agents is administered. The inhibitors of oneor more enzymes of the glutamine metabolism pathway can be administeredas a single treatment or in a series of treatments that continue asneeded and for a duration of time that causes one or more symptoms ofthe cancer to be reduced or ameliorated, or that achieves anotherdesired effect.

The dose(s) vary, for example, depending upon the identity, size, andcondition of the subject, further depending upon the route by which thecomposition is to be administered and the desired effect. Appropriatedoses of a therapeutic agent depend upon the potency with respect to theexpression or activity to be modulated. The therapeutic agents can beadministered to an animal (e.g., a human) at a relatively low dose atfirst, with the dose subsequently increased until an appropriateresponse is obtained.

In non-human animal studies, applications of potential products arecommenced at higher dosage levels, with dosage being decreased until thedesired effect is no longer achieved or adverse side effects disappear.The dosage may range broadly, depending upon the desired effects and thetherapeutic indication. Typically, dosages may be between about 10microgram/kg and 100 mg/kg body weight, preferably between about 100microgram/kg and 10 mg/kg body weight. Alternatively, dosages may bebased and calculated upon the surface area of the patient, as understoodby those of skill in the art.

In some embodiments, the inhibitors of one or more enzymes of theglutamine metabolism pathway can be used in combination and adjunctivetherapies for inhibiting proliferation and/or growth of cancer cellshaving mutated PIK3CA. The phrase “combination therapy” embraces theadministration of the inhibitors described herein and an additionaltherapeutic agent as part of a specific treatment regimen intended toprovide a beneficial effect from the co-action of these therapeuticagents. Administration of these therapeutic agents in combinationtypically is carried out over a defined time period (usually minutes,hours, days or weeks depending upon the combination selected). Thephrase “adjunctive therapy” encompasses treatment of a subject withagents that reduce or avoid side effects associated with the combinationtherapy of the present invention.

A combination therapy is intended to embrace administration of thetherapeutic agents (e.g., inhibitors described herein and/or othertherapeutic agents) in a sequential manner, that is, wherein eachtherapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample, by administering to the subject a single capsule having a fixedratio of each therapeutic agent or in multiple, single capsules for eachof the therapeutic agents. Sequential or substantially simultaneousadministration of each therapeutic agent can be effected by anyappropriate route including, but not limited to, oral routes,intravenous routes, intramuscular routes, and direct absorption throughmucous membrane tissues. The therapeutic agents can be administered bythe same route or by different routes. The sequence in which thetherapeutic agents are administered is not narrowly critical.

Combination therapy also can embrace the administration of thetherapeutic agents described herein in further combination with otherbiologically active ingredients (such as, but not limited to, a secondand different therapeutic agent) and non-drug therapies (such as, butnot limited to, surgery or radiation treatment). Where the combinationtherapy further comprises radiation treatment, the radiation treatmentmay be conducted at any suitable time so long as a beneficial effectfrom the co-action of the combination of the therapeutic agents andradiation treatment is achieved. For example, in appropriate cases, thebeneficial effect is still achieved when the radiation treatment istemporally removed from the administration of the therapeutic agents,perhaps by days or even weeks.

In certain embodiments the inhibitors of one or more enzymes of theglutamine metabolism pathway can be administered in combination at leastone anti-proliferative agent selected from the group consisting of achemotherapeutic agent, an antimetabolite, an antitumorgenic agent, anantimitotic agent, an antiviral agent, an antineoplastic agent, animmunotherapeutic agent, and a radiotherapeutic agent.

The phrase “anti-proliferative agent” can include agents that exertantineoplastic, chemotherapeutic, antiviral, antimitotic,antitumorgenic, and/or immunotherapeutic effects, e.g., prevent thedevelopment, maturation, or spread of neoplastic cells, directly on thetumor cell, e.g., by cytostatic or cytocidal effects, and not indirectlythrough mechanisms such as biological response modification. There arelarge numbers of anti-proliferative agent agents available in commercialuse, in clinical evaluation and in pre-clinical development, which couldbe included in the present invention by combination drug chemotherapy.For convenience of discussion, anti-proliferative agents are classifiedinto the following classes, subtypes and species: ACE inhibitors,alkylating agents, angiogenesis inhibitors, angiostatin,anthracyclines/DNA intercalators, anti-cancer antibiotics orantibiotic-type agents, antimetabolites, antimetastatic compounds,asparaginases, bisphosphonates, cGMP phosphodiesterase inhibitors,calcium carbonate, cyclooxygenase-2 inhibitors, DHA derivatives, DNAtopoisomerase, endostatin, epipodophylotoxins, genistein, hormonalanticancer agents, hydrophilic bile acids (URSO), immunomodulators orimmunological agents, integrin antagonists, interferon antagonists oragents, MMP inhibitors, miscellaneous antineoplastic agents, monoclonalantibodies, nitrosoureas, NSAIDs, ornithine decarboxylase inhibitors,pBATTs, radio/chemo sensitizers/protectors, retinoids, selectiveinhibitors of proliferation and migration of endotheliai cells,selenium, stromelysin inhibitors, taxanes, vaccines, and vincaalkaloids.

In some embodiments, chemotherapeutic agents that may be administered incombination with the inhibitors described herein can include: ABT-263,aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg,bicalutamide, bleomycin, bortezomib, buserelin, busulfan, campothecin,capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil,chloroquine, cisplatin, cladribine, clodronate, colchicine,cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, demethoxyviridin, dexamethasone, dichloroacetate,dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin,estradiol, estramustine, etoposide, everolimus, exemestane, filgrastim,fludarabine, fludrocortisone, fluorouracil and 5-fluorouracil,fluoxymesterone, flutamide, gemcitabine, genistein, goserelin,hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan,ironotecan, lenalidomide, letrozole, leucovorin, leuprolide, levamisole,lomustine, lonidamine, mechlorethamine, medroxyprogesterone, megestrol,melphalan, mercaptopurine, mesna, metformin, methotrexate, mitomycin,mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,paclitaxel, pamidronate, pentostatin, perifosine, PF-04691502,plicamycin, pomalidomide, porfimer, procarbazine, raltitrexed,rituximab, romidepsin, sorafenib, streptozocin, sunitinib, suramin,tamoxifen, temozolomide, temsirolimus, teniposide, testosterone,thalidomide, thioguanine, thiotepa, titanocene dichloride, topotecan,trastuzumab, tretinoin, vinblastine, vincristine, vindesine,vinorelbine, and vorinostat (SAHA). For example, chemotherapeutic agentsthat may be conjointly administered with compounds of the inventioninclude: aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg,bicalutamide, bleomycin, bortezomib, buserelin, busulfan, campothecin,capecitabine, carboplatin, carfilzomib, carmustine, chlorambucil,chloroquine, cisplatin, cladribine, clodronate, colchicine,cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin,daunorubicin, demethoxyviridin, dichloroacetate, dienestrol,diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol,estramustine, etoposide, everolimus, exemestane, filgrastim,fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide,gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide,imatinib, interferon, irinotecan, ironotecan, lenalidomide, letrozole,leucovorin, leuprolide, levamisole, lomustine, lonidamine,mechlorethamine, medroxyprogesterone, megestrol, melphalan,mercaptopurine, mesna, metformin, methotrexate, mitomycin, mitotane,mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,paclitaxel, pamidronate, pentostatin, perifosine, plicamycin,pomalidomide, porfimer, procarbazine, raltitrexed, rituximab, sorafenib,streptozocin, sunitinib, suramin, tamoxifen, temozolomide, temsirolimus,teniposide, testosterone, thalidomide, thioguanine, thiotepa, titanocenedichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine,vindesine, and vinorelbine. In other embodiments, chemotherapeuticagents that may be conjointly administered with compounds of theinvention include: ABT-263, dexamethasone, 5-fluorouracil, PF-04691502,romidepsin, and vorinostat (SAHA).

It will be appreciated that pharmaceutical compositions or formulationsof the inhibitors and/or other therapeutic agents described herein canbe provided in any form, which allows for the composition to beadministered to a patient. For example, the composition may be in theform of a solid, liquid or gas (e.g., aerosol). Other routes ofadministration include, without limitation, oral, topical, parenteral(e.g., sublingually or buccally), sublingual, rectal, vaginal, andintranasal. The term parenteral as used herein includes subcutaneousinjections, intravenous, intramuscular, intrasternal, intracavemous,intrathecal, intrameatal, intraurethral injection or infusiontechniques. The pharmaceutical composition is formulated so as to allowthe active ingredients contained therein to be bioavailable uponadministration of the composition to a patient. Compositions that willbe administered to a patient take the form of one or more dosage units,where for example, a tablet may be a single dosage unit, and a containerof one or more compounds of the invention in aerosol form may hold aplurality of dosage units.

Pharmaceutical compositions can include physiologically acceptablesurface active agents, carriers, diluents, excipients, smoothing agents,suspension agents, film forming substances, and coating assistants, or acombination thereof; and a inhibitor and/or other therapeutic agentdisclosed herein. Acceptable carriers or diluents for therapeutic useare well known in the pharmaceutical art, and are described, forexample, in Remington's Pharmaceutical Sciences, 18th Ed., MackPublishing Co., Easton, Pa. (1990), which is incorporated herein byreference in its entirety. Preservatives, stabilizers, dyes, sweeteners,fragrances, flavoring agents, and the like may be provided in thepharmaceutical composition. For example, sodium benzoate, ascorbic acidand esters of p-hydroxybenzoic acid may be added as preservatives. Inaddition, antioxidants and suspending agents may be used. In variousembodiments, alcohols, esters, sulfated aliphatic alcohols, and the likemay be used as surface active agents; sucrose, glucose, lactose, starch,crystallized cellulose, mannitol, light anhydrous silicate, magnesiumaluminate, magnesium methasilicate aluminate, synthetic aluminumsilicate, calcium carbonate, sodium acid carbonate, calcium hydrogenphosphate, calcium carboxymethyl cellulose, and the like may be used asexcipients; magnesium stearate, talc, hardened oil and the like may beused as smoothing agents; coconut oil, olive oil, sesame oil, peanutoil, soya may be used as suspension agents or lubricants; celluloseacetate phthalate as a derivative of a carbohydrate such as cellulose orsugar, or methylacetate-methacrylate copolymer as a derivative ofpolyvinyl may be used as suspension agents; and plasticizers such asester phthalates and the like may be used as suspension agents.

The pharmaceutical compositions described herein can be administered toa human patient per se, or in pharmaceutical compositions where they aremixed with other active ingredients, as in combination therapy, orsuitable carriers or excipient(s). Techniques for formulation andadministration of the compounds of the instant application may be foundin “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton,Pa., 18th edition, 1990.

The pharmaceutical compositions may be manufactured in a manner that isitself known, e.g., by means of conventional mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating,entrapping or tabletting processes.

Pharmaceutical compositions for use herein may be formulated inconventional manner using one or more physiologically acceptablecarriers comprising excipients and auxiliaries, which facilitateprocessing of the active compounds into preparations, which can be usedpharmaceutically. Proper formulation is dependent upon the route ofadministration chosen. Any of the well-known techniques, carriers, andexcipients may be used as suitable and as understood in the art; e.g.,in Remington's Pharmaceutical Sciences, above.

Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Suitableexcipients are, for example, water, saline, dextrose, mannitol, lactose,lecithin, albumin, sodium glutamate, cysteine hydrochloride, and thelike. In addition, if desired, the injectable pharmaceuticalcompositions may contain minor amounts of nontoxic auxiliary substances,such as wetting agents, pH buffering agents, and the like.Physiologically compatible buffers include, but are not limited to,Hanks's solution, Ringer's solution, or physiological saline buffer. Ifdesired, absorption enhancing preparations (for example, liposomes), maybe utilized.

For transmucosal administration, penetrants appropriate to the barrierto be permeated may be used in the formulation.

Pharmaceutical compositions for parenteral administration, e.g., bybolus injection or continuous infusion, include aqueous solutions of theactive compounds in water-soluble form. Additionally, suspensions of theactive compounds may be prepared as appropriate oily injectionsuspensions. Suitable lipophilic solvents or vehicles include fattyoils, such as sesame oil, or other organic oils such as soybean,grapefruit or almond oils, or synthetic fatty acid esters, such as ethyloleate or triglycerides, or liposomes. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, thesuspension may also contain suitable stabilizers or agents that increasethe solubility of the compounds to allow for the preparation of highlyconcentrated solutions. Formulations for injection may be presented inunit dosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

For oral administration, the inhibitors and/or other therapeutic agentscan be formulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the compounds of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a patient to be treated.Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Dragee cores areprovided with suitable coatings. For this purpose, concentrated sugarsolutions may be used, which may optionally contain gum arabic, talc,polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses. For this purpose, concentratedsugar solutions may be used, which may optionally contain gum arabic,talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, pharmaceutical compositions areconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

Additional therapeutic or diagnostic agents may be incorporated into thepharmaceutical compositions. Alternatively or additionally,pharmaceutical compositions may be combined with other compositions thatcontain other therapeutic or diagnostic agents.

The exact formulation, route of administration and dosage for thepharmaceutical compositions can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl et al. 1975, in “ThePharmacological Basis of Therapeutics”, which is hereby incorporatedherein by reference in its entirety, with particular reference to Ch. 1,p. 1). Typically, the dose range of the composition administered to thepatient can be from about 0.5 to 1000 mg/kg of the patient's bodyweight. The dosage may be a single one or a series of two or more givenin the course of one or more days, as is needed by the patient. Ininstances where human dosages for compounds have been established for atleast some condition, the present invention will use those same dosages,or dosages that are between about 0.1% and 500%, more preferably betweenabout 25% and 250% of the established human dosage. Where no humandosage is established, as will be the case for newly-discoveredpharmaceutical compounds, a suitable human dosage can be inferred fromED₅₀ or ID₅₀ values, or other appropriate values derived from in vitroor in vivo studies, as qualified by toxicity studies and efficacystudies in animals.

Example

In this Example we show that PIK3CA mutations render cancers, such ascolorectal cancers (CRC), more sensitive to glutamine deprivation byup-regulation of glutamate pyruvate transaminase 2 (GPT2), an enzymeinvolved in glutamine metabolism. We further show that mutant p110αincreases GPT2 gene expression through an AKT-independent signalingpathway. Moreover, we show that aminooxyacetate (AOA), and EGCG,compounds that inhibit enzymatic activity of antitransferases, as wellas BPTES andCB-839, compounds that inhibit enzymatic activity ofglutaminose, can suppress xenograft tumor growth of CRCs with PIK3CAmutations, but not CRCs with wildtype (WT) PIK3CA. These resultsdemonstrate that reprogramming glutamine metabolism is crucial for theoncogenic function of PIK3CA mutations and that targeting glutaminemetabolism can be an effective approach to treating cancer patientsharboring mutations of this gene.

Methods Cell Culture

Colorectal cancer (CRC) cell lines, HCT116, DLD1, RKO, HT29, SW480 andLOVO, were cultured in Mccoy's 5A medium containing 10% fetal bovineserum (FBS) as described previously. Mccoy's 5A (Cat No. SH30200), fetalbovine serum (Cat No. SH30910), and Glutamine-free DMEM (with 4.5 g/Lglucose, without pyruvate, Cat No. SH30081) were obtained from Hyclone.Dialysed FBS (dFBS, Cat No. 26400) and Glutamine, Glucose free DMEM (CatNo. A14430) were obtained from Gibco (Invitrogen).

Chemicals, siRNAs, Plasmids and Antibodies

L-Glutamine, L-Glutamine-₁₃C₅, D-Glucose, dimethyl α-Ketoglutarate, andAminooxyacetate (AOA) were purchased from Sigma. siRNAs of ATF4(SI03019345, SI04236337) and PDK1 (SI00301140, SI00301154) werepurchased from Qiagen. siRNAs of RSK2 (J-003026-10, J-003026-12) werepurchased from Dharmacon. siRNAs of USP8 (SR306014A, SR306014B) werepurchased from Origene. shRNAs of GPT2 (TRCN0000035025, TRCN0000035026)were purchased from Sigma. Adeno-ATF4 virus was made as described in.cDNAs of GPT2, ATF4 and β-TrCP were purchased from Addgene. GPT2 ORF wassubcloned into pCMV-3Tag1A vector with HindIII and SaII. Then Flag-GPT2sequence was PCR out and subcloned into pCDNA3.1zeo with KpnI and XbaI.Flag tagged ATF4 expression vector was constructed by sub-cloning ATF4ORF into the pCMV-3Tag1A vector with BamHI and XhoI. ATF4 S219A andS245A mutant constructs were made by Quick-change Site-DirectedMutagenesis kit (Agilent Technologies). Plasmids were transfect intocells with Lipofectamine 3000 (Invitrogen) according to manufacturerinstruction. For transient expression, cells were lysed 72 hours aftertransfection. For stable expression (FLAG-GPT2 expression), cells wereselected with 0.5 mg/ml Zeocin (Invitrogen) for 7 days. All primers andantibodies used in this Example are listed in Tables 1 and 2.

TABLE 1 Primers used Targeting primers Left Arm forwardGGGAAAG/ideoxyU/GATGAGTCTGTCGGTGTTTGTG D933A reverseAAAGCTATATGAAACAGCTTTCAAA D933A forwardTTTGAAAGCTGTTTCATATAGCTTTTGGACACTTTTTGG ATC Left Arm reverseGGAGACA/ideoxyU/TTTTGTGTTTTTAATTGCTCGAGC p110α D933A Right ArmGGTCCCA/ideoxyU/CTGGCTGCTCTATTAGAAACAATC forward Knock-In Right ArmGGCATAG/ideoxyU/GATGTTGACATGGATGTGGTGA vector reverse Screening forwardCTGCAGTTCAACAGCCACAC Screening reverse CAGGGAAATGCAAATTAAAACCCre forward GTAAAGGAGCCCAAGAATGC Cre reverse GCCAACATTTATTATTTTGAAATTGSubcloning primers GPT2 Forward CCCAAGCTTATGCAGCGGGCGGCGGCGC pCMV-Reverse ACGCGTCGACTCACGCGTACTTCTCCAGGAAG 3Tag1A Flag-GPT2 ForwardCGGGGTACCGCCACCATGGATTACAAGGATGACGACG pCDNA3.1zeo ReverseTGCTCTAGATCACGCGTACTTCTCCAGGAAG FLAG-ATF4 ForwardCGCGGATCCATGACCGAAATGAGCTTCCTGAG pCMV- ReverseCCGCTCGAGCTAGGGGACCCTTTTCTTCC 3Tag1A Myc-β-TrCP ForwardGGTCCCA/ideoxyU/TGGACCCGGCCGAGGCGGTG pCMV-Tag2 ReverseGGCATAG/ideoxyU/TCTGGAGATGTAGGTGTATG GPT2 ForwardCGGGGTACCCTGGGGAAGACTTTTACCTA promoter pGL3 ReverseGGAAGATCTCCACAGCCGCATCCCCGCGC Mutagenesis primers FLAG-ATF4 ForwardCTTCAGATAATGATGCTGGCATCTGTATGAGC S219A ReverseGCTCATACAGATGCCAGCATCATTATCTGAAG FLAG-ATF4 ForwardCAGGGGCTCTCCAAATAGGGCGCTCCCATCTCCAGGTG S245A TTC ReverseGAACACCTGGAGATGGGAGCGCCCTATTTGGAGAGCC CCTG GPT2 ForwardCGGAAGTGATGGAGGTCGTTGCGCTAATGGAGTGGTC promoter GGGAAAAC Mut1 ReverseGTTTTCCCGACCACTCCATTAGCGCAACGACCTCCATC ACTTCCG GPT2 ForwardGCACCGTGTGGCCTTGGAGTTGCGCTACTCGGGGCGAT promoter GACTGCAC Mut2 ReverseGTGCAGTCATCGCCCCGAGTAGCGCAACTCCAAGGCC ACACGGTGC RT-PCR primers SLC1A5Forward CATCATCCTCGAAGCAGTCA Reverse CTCCGTACGGTCCACGTAAT GLS1 ForwardTGCATTCCTGTGGCATGTAT Reverse TTGCCCATCTTATCCAGAGG GLS2 ForwardGACTTCTCAGGGCAGTTTGC Reverse TGGTTGAACTGCACAGCATC GPT1 ForwardATGGCCTCGAGCACAGGTGAC Reverse CAGCACCGTCACGATGGCATC GPT2 ForwardCTTTCTCCTGGCTGATGAGG Reverse TAACCACACTCGCCCATGTA GOT1 ForwardACCTGGGAGAATCACAATGC Reverse GCGGCTGTGCCCGCCGGTGC GOT2 ForwardCAATGGCTGCAAGAAGTGAA Reverse GGCTTTAGCCCTGTGAAACA GLUD1 ForwardCACACGCCTGTGTTACTGGT Reverse CTCCAAACCCTGGTGTCATT GAPDH ForwardGGAAATCCCATCACCATCT Reverse TGTCGCTGTTGAAGTCAGA ATF4 ForwardCCAACAACAGCAAGGAGGAT Reverse AGTGTCATCCAACGTGGTCA

TABLE 2 Antibodies Antibodies Application Company Catalog number GPT2 IBProteintech Group 16757-1-AP GLS1 IB Proteintech Group 20170-1-AP ATF4IB, IP Santa Cruz sc-200 HA IB Santa Cruz sc-805 GAPDH IB Santa Cruzsc-25778 c-myc IB Santa Cruz sc-40 PUMA IB Cell Signaling 4976S p-FoxoIB Cell Signaling 9464S RSK2 IB Cell Signaling 5528S p-eIF2α IB CellSignaling 3398S eIF2α IB Cell Signaling 9722 Ubiquitin IB Cell Signaling3936 USP8 IB Cell Signaling 8728 USP7 IB Cell Signaling 4833 USP1 IBCell Signaling 8033 USP2 IB Cell Signaling 8036 USP9X IB Cell Signaling5751 USP10 IB Cell Signaling 8501 USP14 IB Cell Signaling 8159 USP18 IBCell Signaling 4813 Cleaved PARP IB Cell Signaling 9544 Cleaved Caspase3IB Cell Signaling 9664 Foxo1 IB Millipore 05-1075 pATF4 S245 IB Abcamab28830 PDK1 IB Abcam ab52893 FLAG IB Sigma F1804

Quantitative Real Time PCR

One μg of total RNA was used for Reverse transcription by SuperscriptFirst-Strand kit (Invitrogen). cDNA was used for real time PCR. Taqmanassay system was used for qRT-PCR using GPT2 (Hs00370287, Appliedbiosystems) probes with IQ super mix (170-8860, Bio-Rad). Expressionlevels of GPT2 in each tumor were normalized to that of B2M. Mutationstatus of the human CRC specimens are listed in Table 3.

TABLE 3 Tumor Samples of Quantitative Real-Time PCR Tumors PIK3CAmutation Tumors Mutation in PI3K pathway 435X PIK3CA E545K 560X WT 507XPIK3CA E545K 569X WT 533X PIK3CA H1047R 492X WT 511X PIK3CA Q546K 452XWT 587X PIK3CA H1047R 493X WT 480X PIK3CA R38C 566X WT 579X PIK3CAH1047R 586X WT 823X PIK3CA H1047R 559X WT X841 PIK3CA H1047R 464X WTX850 PIK3CA E542K 502X WT

Somatic Gene Targeting

The PIK3CA D933A targeting vector was constructed with USER system, andtargeted cells were generated as described previously. Briefly, vectorarms were created by PCR from genomic DNA using HiFi Taq (Invitrogen)and validated by sequencing prior to viral production and infection.DLD1 Mutant cells were infected with rAAV viruses. Stable G418-resistentclones were then selected for PCR screening as reported. Targeted cloneswere genotyped by RTPCR and sequencing. Detailed information onconstruction of targeting vectors and targeted cells is available uponrequest.

Immunoblotting and Immunoprecipitation

Cells were lysed in RIPA buffer [10 mM Tris (pH 7.4), 150 mM NaCl, 5 mMEDTA (pH 8.0), 0.1% SDS, 1% Triton-X100, 1 mM DTT, 1 mM PMSF, completeProtease Inhibitor Cocktail tablet (Roche); supplemented withphosphatase inhibitors (1 mM Na₃VO₄, 50 mM NaF, 1 mM β-glycerophosphate,20 mM sodium pyrophosphate)]. Lysates were then cleared bycentrifugation at 14,000 rpm for 10 min and protein concentration insupernatants was determined by the BCA protein assay kit (Pierce). Equalamounts of total protein were used for immunoblotting. Forimmunoprecipitation (IP), cells were lysed as mentioned above. Cleanedcell lysate incubated with antibody for one hour, and then protein Aand/or protein G for one hour. Protein A/G beads were washed with lysisbuffer three times, and then boiled with SDS-loading buffer followedwith immunoblotting.

Luciferase Reporter Assay

A 1.5 kb promoter region of GPT2 was subcloned into pGL3 vector(Promega) to obtain pGL3-GPT2 promoter-LUC plasmid. pGL3-GPT2promoter-LUC was co-transfect with pCMV-ATF4 and internal controlβ-galactosidase expressing pCH110 (abbreviation as pCH110 β-gal) orRenilla luciferase expressing pRL (Promega). 48 hours aftertransfection, cells were harvest for Luciferase assay according tomanufacturer instruction (Promega). Luminescence was measured withEnVision 2103 Multilabel Plate Readers (PerkinElmer). β-galactosidaseactivity was measured with β-Gal assay kit (Invitrogen). Mutation ofGPT2 promoter was generated with Quickchange kit (Agilent Technologies)and primers is in Table 2. The uORFATF4 plasmid is a kind gift from Dr.Ron Wek at Indiana University.

Cell Proliferation Assay

Cells were plated in 96-well plates at 2000 cells per well, 24-wellplates at 1×10₄ cells per well, 6-well plates at 2×10⁵ cells per well incomplete DMEM [20 mM Glucose, 2 mM Glutamine, 10% dialysed fetal bovineserum (dFBS), Invitrogen]. After 24 hours, cells were washed with PBS,and changed to either glutamine deprived DMEM (with 20 mM Glucose) orglucose deprived DMEM (with 2 mM Glutamine) containing 10% dFBS. Cells(including floating cells in medium) were collected and counted byTrypan-Blue exclusive assay.

Flow Cytometry

Cells were fixed with methanol and then incubated at 37° C. for 30 minin 5% normal goat serum diluted in PBS. Propidium iodide (PI) solutionwas used to stain cells at 4° C. for 1 hour. Cells were analyzed on anEpics XL flow cytometer. WinMDI2.9 was used for data analysis. Celldebris and aggregates were excluded on PI gating. Percentages of sub-G1,G1, S and G2/M populations were determined by histograms generated byWinDI2.9.

Gene Silencing

Plasmids expressing shRNAs were transfect into cells. Two dayspost-transfection, cells were selected with 1 μg/ml of puromycin for 7days. Puromycin resistance cells were collected, amplified, andanalyzed. For genes silenced by siRNAs, siRNAs were transfect into cellswith Lipofectamine 3000. Three days post-transfection, cells wereharvested for further analyses.

Ubiquitination Assay

Cells were pre-treated with 5 μM of MG132 for 6 hours and cell lysateswere immunoprecipitated with antibodies against either ATF4 or FLAG.Beads were washed with washing buffer [10 mM Tris (pH 7.4), 1 M NaCl, 1mM EDTA (pH 8.0), 1% NP-40] for 3 times. The immunocomplexes wereresolved in SDS-PAGE gels for Western blot analyses.

Polysome Profile Analysis

Three tumors (˜250 mm3 size) of each genotype were snap-frozen in liquidnitrogen, pulverized and lysed in 1000 μl of lysis buffer (50 mMHEPES-KOH (pH 7.4), 5 mMMgCl2, 250 mMKCl, 2% TritonX-100, 8.5% sucrose,100 μg/ml cycloheximide, 1 mM DTT, 200 units/ml RNase inhibitor(RNaseOUT, Invitrogen), EDTA-free protease inhibitor (Roche AppliedScience) and 10 mM ribonucleoside vanadyl complex (New EnglandBiolabs)), kept on ice for 20 min, and then passed 15 times through a23-gauge needle. Lysates were spun at 14,000 rpm for 15 min, andsupernatants were collected. Approximately 10-15 A units (260 nm) oflysates were layered over 10-50% cold sucrose gradients in buffer (50 mMHEPES-KOH (pH 7.4), 5 mM MgCl2, 250 mM KCl). Gradients were centrifugedat 17,000 rpm in a BeckmanSW28 rotor for 15 h at 4° C. Aftercentrifugation, 12 fractions (1.2 ml/fraction) were collected. RNA fromeach fraction was isolated using TRIzol LS reagent (Invitrogen), and anequal volume of RNA from each fraction was used for cDNA synthesis. Therelative quantities of specific mRNAs were measured by quantitativeRT-PCR (RT-qPCR).

Xenograft Study

Animal experiments were approved by the Case Western Reserve UniversityAnimal Care and Use Committee. As described in, 3 million cells wereinjected subcutaneously into the flanks of 4 to 6-week-old femaleathymic nude mice. Mice were randomly assigned into treatment groups (5mice/group). When average tumor volume reached 100 mm3, mice weretreated with vehicle control, 5 mg/kg or 10 mg/kg of AOA every day perIP (intraperitoneal) injection. Tumor volumes were measured with anelectronic caliper and calculated as length×width²/2.

Metabolic Assays and Stable Isotope Tracing

A million cells were plated in each T25 flask. When reached at 70%confluency, cells were washed with PBS twice and changed to mediumcontaining 2 mM of [¹³C₅-]Glutamine. Cells were grown in medium foreither 2 hours for enrichment assay or 24 hours for relative abundanceassay. Cells were then quenched and harvested with 1 ml pre-chilled(−80° C.) methanol. Five μM of heptadecanoic acid, 2.5 μM of[3,3,4,5,5,5⁻²H6]4-hydroxypentanoate and 2.5 μM of[2,2,3,3,4,4,5,5,6,6,7,7,7⁻²H13]heptanoate were added as internalstandards. Metabolites were extracted by homogenization and sonicationon ice. Cell debris was discarded by centrifuge at 14,000 rpm, 15 minsat 4° C. The supernatant was dried with nitrogen gas. TBDMS(MTBSTFA+TBDMCS, REGIS Technologies): Acetonitrile (2:1) was used forderivatization of metabolites at 60° C. for 1 hour. Samples (1 μl) wereinjected into GC-MS (Agilent Technologies) for metabolite profiling. Forenrichment analyses, total pool of each metabolite was considered as100%. M (0, 1, 2, 3, ect.) indicated the number of ¹³C labelled carbon.Enrichment indicates percentage of each isotopomer to total pool. Forrelative abundance analyses, each ¹³C labelled isotopomer ornon-labelled metabolite was normalized to an internal standard with sameresponse time range in GC-MS.

Assays of ATP/ADP and NADH/NAD

The amounts of ATP and ATP/ADP ratios were measured with an ADP/ATPratio assay kit (Abcam) according to the manufacturer's instructions.The amounts of ATP were determined by an ATP standard curve, andnormalized to the protein concentrations. The amounts of NADH andNADH/NAD ratios were measured with a NAD/NADH quantitation colorimetrickit (BioVision). The NADH concentrations were determined by a NADHstandard curve, and normalized to the protein concentrations.

Results PIK3CA Mutations Render CRC Cells Dependent on Glutamine

Most PIK3CA mutations are clustered in two hot spots: H1047R in thekinase domain and E545K in the helical domain. We set out to determinewhether PIK3CA mutations reprogram cell metabolism in CRCs. The CRC cellline HCT116 harbors a heterozygous H1047R mutation, whereas DLD1 CRCcells express a heterozygous E545K mutation (FIG. 2A). We exploitedisogenic derivatives of these cell lines with either the WT or mutantalleles of PIK3CA knocked out (FIG. 2A). The clones in which the mutantallele had been disrupted and the wild-type allele was intact werecalled “wild-type” (WT, FIG. 2A), whereas the clones in which the WTallele had been disrupted and the mutant allele was intact were called“mutant” (Mut, FIG. 2A). As reported previously, the parent cells andknockout clones grew at similar rate under normal conditions in thepresence of both glucose and glutamine (FIG. 2B). However, both parentalcells and the PIK3CA mutant clones grew considerably more slowly inmedium without glutamine than did PIK3CA WT clones (FIG. 2B). Thisrelative sensitivity to glutamine deprivation was observed in bothHCT116 and DLD1 cell lines containing mutant PIK3CA genes (FIG. 2B).Glutamine deprivation induced more apoptotic cells in the mutant clonesthan in 7 the WT clones as assayed by percentages of sub-G1 cells andamounts of cleaved PARP (FIGS. 2(C-D)). In contrast, none of these celllines showed differential sensitivity to deprivation of glucose (FIG.2B). To determine what we observed with the isogenic cell lines weregeneralizable, we tested glutamine sensitivity in two CRC cell lineswith PIK3CA mutations [RZKO (containing a PIK3CA H1047R mutation) andHT29 (containing a PIK3CA P449T mutation)] and two CRC cell lines withWT PIK3CA (SW480 and LOVO). As shown in FIG. 2E, glutamine deprivationinduced significantly more apoptotic cells in the two PIK3CA mutant celllines than in the two WT PIK3CA cell lines. Consistently, when deprivedof glutamine, relative survival rates of the two WT PIK3CA cell lineswere higher than those of the two PIK3CA mutant cell lines. Takentogether, the data suggest that PIK3CA mutations render CRC cells moredependent on glutamine for optimal growth.

The Up-Regulation of GPT2 by PIK3CA Mutations Renders CRC CellsDependent on Glutamine

To determine whether PIK3CA mutations regulate the transcription ofenzymes involved in glutamine metabolism, we performed serial analysisof gene expression (SAGE) on the isogenic cell lines in the Appendix.Interestingly, the expression levels of mitochondrial glutamate pyruvatetransaminase GPT2, which converts glutamate to α-KG, were up-regulatedin both HCT116 and DLD1 PIK3CA-mutant clones compared to the WT clones.This observation was confirmed by both RT-PCR and Western blot analysesof the clones (FIGS. 3(A-B)). However, the cytosolic glutamate pyruvatetransaminase GPT1 was not expressed or expressed at only an extremelylow levels in the clones (FIG. 3A). None of the other enzymes primarilyinvolved in glutamine metabolism including GOT1, GOT2, Glud1, GLS1 andGLS2, or the glutamine 8 transporter SLC1A5, exhibited any differentialexpression among the PIK3CA mutant and WT clones (FIG. 3A).

We next test if GPT2 expression is up-regulated in CRCs specimens withPIK3CA mutations. We thus measured GPT2 RNA levels by qRT-PCR in 21human CRC patient tumors that we performed whole-exon sequencingpreviously (10 tumors with PIK3CA mutations and 10 tumors with nomutations in the PIK3CA pathway). As shown in FIG. 3C, expression levelsof GPT2 were significantly higher in the tumors with PIK3CA mutationthan in these tumors with WT PIK3CA.

To determine whether the up-regulation of GPT2 makes PIK3CA mutant cellsdependent on glutamine, we knocked down GPT2 in the HCT116 mutant cloneusing two independent shRNAs, then grew the cells in normal medium ormedium without glutamine. Compared to cells with a control shRNA,knockdown of GPT2 made the PIK3CA mutant cells less sensitive toglutamine deprivation as assayed by relative cell growth (FIG. 3D) andcell apoptosis, even though the GPT2 knockdown cells grew more slowlyunder normal growth conditions. In contrast, knockdown of GPT2 in theHCT116 PIK3CA WT clone had no effect on their sensitivity to glutaminedeprivation or proliferation under normal culture conditions (FIG. 3E).Conversely, overexpression of GPT2 in the WT clone made it sensitive toglutamine deprivation. In aggregate, these data demonstrate that PIK3CAmutations render colorectal cancer cells more sensitive to glutaminewithdrawal through an up-regulation of GPT2.

An Aminotransferase Inhibitor Suppresses the Growth of PIK3CA-Mutant CRCCell Lines In Vivo

AOA inhibits the enzymatic activity of aminotransferases includingGPT220. As shown in FIG. 10 a, both HCT116 and DLD1 PIK3CA mutant cloneswere more sensitive to AOA treatment than the WT clones in tissueculture. Moreover, AOA significantly inhibited the growth of HCT116 andDLD1 mutant clones when xenografted into nude mice (FIGS. 4(A-B)). Incontrast, AOA had no effect on isogenic PIK3CA WT xenograft tumors(FIGS. 4(A-B)), although those tumors grew more slowly than their mutantcounterparts in the absence of AOA (FIGS. 4(A-B)). To test whether ourobservations with the genetically engineered cell lines weregeneralizable, we expanded our xenograft study to a panel of CRC celllines. AOA inhibited xenograft tumor growth of four PIK3CA-mutant CRCcell lines [HCT116 (parental cells), DLD1 (parental cells), RKO andHT29, FIG. 4C]. In contrast, AOA had no effect on xenograft tumor growthof two WT PIK3CA CRC cell lines (SW480 and LOVO, FIG. 4D). No weightloss was observed for the mice that were treated with AOA, suggestingthat the doses of AOA used in the experiments had minimal toxicity.Consistent with the hypothesis that up-regulation of GPT2 by PIK3CAmutations renders colorectal cancer cells dependent on glutamine, GPT2protein levels were higher in the PIK3CA mutant lines (HCT116, DLD1, RKOand HT29) than in the PIK3CA WT cell lines (SW480 and LOVO).

ATF4 Regulates Transcription of GPT2

Given that p110α is not a transcription factor, a key question raised bythese data is how mutant p110α transduces the signals that activate GPT2transcription. To address this question, we evaluated ATF4, as recentstudies reported that ATF4 is involved in glutamine metabolism.Interestingly, ATF4 protein levels mirrored GPT2 protein levels in thePIK3CA mutant and WT clones (FIG. 5A). This correlation was maintainedin xenograft tumors (FIG. 5B). In contrast, GLS protein levels wereuncorrelated with ATF4 protein levels in the same cell lines (FIG. 5A).Because ATF4 induces expression of pro-apoptotic BH3-only proteins PUMAand Noxa in neuroblastoma cells, we also measured levels of these twoproteins in the PIK3CA WT and mutant clones. However, PUMA and Noxaprotein levels were not correlated with ATF4 levels (FIG. 5A),suggesting that the regulation of the two pro-apoptotic proteins by ATF4may be celltype specific or controlled by other factors.

As shown in FIG. 5C, overexpression of ATF4 in the HTC116 PIK3CA WTclone increased GPT2 protein levels in a dose-dependent manner.Conversely, knockdown of ATF4 in the HCT116 PIK3CA mutant clone by twodifferent siRNAs reduced both mRNA and protein levels of GPT2 (FIG. 5D).In contrast, knockdown of ATF4 did not affect the expression of otherenzymes involved in glutamine metabolism, including SLC1A5, GLS1, GOT1,GOT2 and GLUD1 (FIG. 5A). Similar results were observed in both HCT116and DLD1 PIK3CA mutant clones Importantly, as in the case with GPT2, theknockdown of ATF4 made PIK3CA mutant cells more resistant to glutaminedeprivation as assessed by both cell survival and cell death (FIG. 5E),even though the ATF4 knockdown cells grew more slowly than the controlcells in the presence of glutamine. These results suggest that themutant p110α-ATF4-GPT2 axis regulates glutamine metabolism.

To determine whether ATF4 activates GPT2 gene transcription directly, wecloned a 1.5 kb genomic upstream of the transcription start site of theGPT2 gene into a luciferase reporter 11 plasmid. As shown in FIG. 4 f,knockdown of ATF4 in the HCT116 PIK3CA mutant clone reducedtranscriptional activity of the GPT2 reporter (FIG. 5F). Examining theDNA sequences in the 1.5 kb genomic DNA fragment of GPT2, we found twosequences matching ATF4 consensus binding sites (FIG. 5G). Mutatingeither or both of the two candidate sites significantly diminishedATF4-mediated transcriptional activity (FIG. 5H).

Mutant p110α Stabilizes the ATF4 Protein

We next sought to determine how ATF4 is differentially regulated inPIK3CA-mutant and WT cells. We first showed that ATF4 mRNA levels weresimilar in isogenic PIK3CA mutant and WT cell clones. The ATF4 proteinis known to be induced by stress through phosphoelF2α(p-eIF2α)-dependent translation initiation of upstream open readingframes (uORF). To investigate whether mutant p110α up-regulates ATF4protein levels through this mechanism, we examined p-eIF2α levels in thePIK3CA mutant and WT clones. Similar levels of p-eIF2α were observed inthe PIK3CA mutant and WT clones. This result was consistent with theuORF reporter assays indicating that the upstream ORF initiationactivity of ATF4 was similar in the HCT116 PIK3CA mutant and WT cells.Moreover, polysome profiling of ATF4 mRNA showed no significantdifference between HCT116 PIK3CA mutant and WT clones. We also testedwhether mutant p110α affected ATF4 protein stability by regulating itsubiquitination. As shown in FIGS. 6(A-B), the ubiquitination levels ofboth endogenous and ectopically overexpressed ATF4 were much higher inthe HCT116 PIK3CA WT clone than in the mutant cells. As expected,exposure to a PI3K inhibitor (LY294002), a PI3K/mTOR dual inhibitor(DEZ235) and a PDK1 inhibitor (GSK2334470) each reduced ATF4 proteinlevels (FIG. 6C). Surprisingly, neither an AKT inhibitor (GSK690693) noran inhibitor (CHIR-99021) of GSK3β(a known downstream effector of AKT)had any effect on ATF4 protein levels (FIG. 6C). In accord with this,expression of a constitutively active form of AKT1 (myristylated-AKT1)in the PIK3CA WT clone did not affect ATF4 or GPT2 proteins levels. As acontrol for these experiments, we found that myristylated-AKT1 didincrease the phosphorylation level of FOXO1, a well-known AKT kinasetarget.

Interestingly, ATF4 is reported to be a substrate of RSK2. Although itis not as well-known an effector of PIK3CA as AKT, PDK1 also regulatesthe RSK2 kinase. Indeed, both a pan RSK inhibitor (BI-D1870) and a moreselective RSK2 inhibitor (FMK) reduced ATF4 protein levels in the HCT116PIK3CA mutant clone (FIG. 6C). Therefore, these data suggest ap110α-PDK1-RSK2 pathway that regulates ATF4 protein levels (FIG. 6D).

To confirm the results obtained with the inhibitors, we firstoverexpressed p110α E545K and H1047R mutant constructs in the HCT116PIK3CA WT clone. We found that overexpression of mutant p110α increasedboth ATF4 and GPT2 protein levels (FIG. 6E). We then attempted toascertain whether the lipid kinase activity of p110α is required for themutant p110α signaling pathway to stabilize the ATF4 protein. For thispurpose, we knocked in a D933A mutation that inactivates its lipidkinase activity on top of the E545K mutant allele into the DLD1 PIK3CAmutant clone. FIG. 6F shows that the protein levels of both ATF4 andGPT2 were reduced in the double mutant clones. As expected, AKTphosphorylation levels were also 13 reduced in the double mutant cellsImportantly, the kinase inactivation mutation rendered the DLD1 PIK3CAE545K mutant clone less sensitive to glutamine deprivation (FIG. 6G).Moreover, knockdown of either PDK1 or RSK2 in the HCT116 PIK3CA mutantclone reduced ATF4 and GPT2 protein levels (FIGS. 6(H-I)).

Phosphorylation of ATF4 at S245 by RSK2 Recruits Deubiquitinase USP8 toProtect ATF4 from Degradation

RSK2 is a serine/threonine kinase that phosphorylates ATF4 at the serine245 residue (S245). Knockdown of RSK2 in HCT116 PIK3CA mutant clonereduced levels of pS245 ATF4 (FIG. 7A). Therefore, we hypothesized thatphosphorylation of ATF4 at S245 by the mutant p110α-PDK1-RSK2 signalingaxis stabilizes ATF4. To test this hypothesis, we first examined ATF4S245 phosphorylation in the HCT116 PIK3CA WT and mutant clones. Asexpected, levels of pS245 ATF4 were higher in the mutant clone than inthe WT clone (FIG. 7B). Second, compared to the expression of a WT ATF4construct, the expression of an unphosphorylatable ATF4 S245A mutantconstruct in the HCT116 PIK3CA mutant clone resulted in less ATF4protein (FIG. 7C). In contrast, the ATF4 S219A mutant that abolishes thebinding of ATF4 to β-TrCP1, an ubiquitin E3 ligase of ATF421, generatedmore protein than the WT counterpart (FIG. 7C). Consistent with ourhypothesis that phosphorylation of ATF4 at the S245 residue stabilizesit, the ubiquitination levels of the ATF4 S245A mutant were higher thanthat of the WT protein (FIG. 7D). These data led us to postulate thatphosphorylation of ATF4 S245 by RSK2 either reduces its binding affinityto an ubiquitin E3 ligase or recruits a deubiquitinase, therebyprotecting ATF4 from degradation. To this end, we first tested thebinding of WT and S245A mutant ATF4 to β-TrCP1. However, both WT and themutant ATF4 bound to a similar amount of β-TrCP1. We 14 then turned ourattention to deubiquitinases and tested the binding of the WT ATF4 andS245A mutant to eight USPs (USP1, USP2, USP7, USP8, USP9X, USP10, USP14and USP18). Among the USPs tested, only USP8 exhibited differentialbinding to WT vs mutant ATF4 (FIG. 7E). Consistent with our hypothesis,the ATF4 S245A mutant bound to less USP8 than WT ATF4 (FIG. 7E).Knockdown of UPS8 by two independent siRNAs in the HCT116 PIK3CA mutantclone resulted in reduced ATF4 protein levels (FIG. 7F) and increasedATF4 ubiquitination (FIG. 7G).

TCA Cycle Metabolites are Higher in PIK3CA-Mutant Clones than inIsogenic WT Clones

Both the HCT116 and DLD1 mutant clones consumed more glutamine thantheir WT counter parts. Glutamine is converted to α-KG to replenish theTCA cycle. Because GPT2, an enzyme that converts glutamate to α-KG, isup-regulated in PIK3CA mutant CRC cells (FIG. 3A), we profiled TCA cycleintermediates in the paired isogenic lines. PIK3CA mutant and WT cellswere exposed to 2 mM of [¹³C₅-]glutamine in the presence of glucose for2 hours and the enrichment of the isotope-labeled TCA cycleintermediates was measured by GCMS. All of the measured ¹³C-labeled TCAcycle metabolites, including α-KG, succinate, fumarate, malate andcitrate, were significantly higher in the PIK3CA mutant clones than inthe WT clones. Conversely, the unlabeled TCA cycle intermediates weresignificantly lower in the PIK3CA mutant clones than in the WT clones.Similar results were observed with clones derived from both HCT116 andDLD1. We next examined the steady state of glutamine-derived TCA cycleintermediates by culturing the HCT116 and DLD1 PIK3CA WT and mutantclones in [¹³C₅-]glutamine-containing medium for 24 hours. The resultsshowed that: (i) the majority of the TCA cycle intermediates 15 werederived from glutamine in both PIK3CA mutant and WT cells (FIG. 8A),consistent with the “Warburg effect”; and (ii) compared to the WTclones, the amounts of α-KG and citrate were significantly higher in themutant clones (FIG. 8A).

A major product of the forward TCA cycle is NADH, which couples withoxidative phosphorylation to generate ATP. We therefore measured theamounts of ATP and NADH in the PIK3CA WT and mutant clones. In thepresence of glutamine, the amounts of ATP and NADH were significantlyhigher in both the HCT116 and DLD1 mutant clones than in their WTcounterparts (FIGS. 8(B-C)). The ratios of NADH/NAD were alsosignificantly higher in the mutant clones than in the WT clones.Although not statistically significant, the ATP/ADP ratios appeared tobe higher in the mutant clones. However, under glutamine deprivation,the amount of ATP and the ATP/ADP ratio were significantly lower in themutant clones than in the WT clones (FIG. 8), whereas the amounts ofNADH and the ratios of NADH/NAD were similar in the mutant and WT clones(FIG. 8C).

The Addition of α-KG Rescues Survival of PIK3CA Mutant Cells Deprived ofGlutamine

The results described above show that the generation of α-KG fromglutamine to replenish the TCA cycle was critical to the survival of thePIK3CA mutant cells. To test this suggestion, we deprived the HCT116PIK3CA mutant cell of glutamine and then supplemented the cells with 4mM α-KG. When deprived of glutamine, less than 8% cells survived after 3days (FIG. 8D). In contrast, the addition of α-KG increased cellsurvival to ˜60%.

AOA May Synergize with 5-FU to Inhibit Xenograft Tumor Growth

To determine if AOA can enhance the efficacy of existing colorectalcancer drugs, we tested combination of AOA (10 mg/kg) with 5-FU,irinotecan, oxaliplatin or regorafenib on xenograft tumors establishedHCT116. As shown in FIG. 10, combination of AOA with 5-FU at a dose of10 mg/kg or 20 mg/kg appeared to have synergistic tumor inhibitoryeffect, although higher doses of 5-FU need to be tested to determine thesynergy. In contrast, none of the other drug showed any combinationaleffect with AOA.

IC₅₀ of AOA to GPT2

We have demonstrated that AOA preferentially inhibits xenograft tumorgrowth of colorectal cancers with PIK3CA mutations. However, AOA ispan-transaminase inhibitor. As shown in FIG. 11A, we have developed aGPT2 enzymatic assay that can be easily adapted for high-throughputscreening. The IC₅₀ of AOA to GPT2 is 980 nM (FIG. 11B). Therefore, morepotent and specific GPT2 inhibitor can be developed. Moreover, AOAtreatment at a dose of 10 mg/kg, which resulted in significant growthinhibition of xenograft tumors established from colorectal cancersharboring PIK3CA mutations, only inhibited GPT activity by ˜30% in theAOA treated tumors as assayed by the amounts of α-KG (a product of GPT2,FIG. 12C). Together, our data suggest that a more potent and specificGPT2 inhibitor should be more effective and less toxic than AOA.

Combination of CB-839 with 5-FU Shrinks PIK3CA Mutant Xenograft Tumors

As discussed above, CB-839 is a potent glutaminase inhibitor that blocksthe first step of glutamine metabolism. CB-839 is currently in phase Iclinical trials for several cancer types, but not colorectal cancer. Weset out to determine if CB-839 alone or in combination with 5-FUinhibits xenograft tumor growth of a PIK3CA mutant CRC. As shown in FIG.12, the CB-389 and 5-FU combination treatment induced tumor regression(2 out of 10 tumors regressed after two weeks of treatment, 3 moretumors regressed after three weeks of treatment, and other tumorsstopped growing after two weeks of treatment). In contrast, althoughCB-839 or 5-FU alone inhibited tumor growth to various extents, neitherinduced tumor regression (FIG. 12B). In summary, these preliminaryresults provide a strong rationale for clinical trials of combinationtherapy of CB-839 with 5-FU in CRCs with PIK3CA mutations.

PIK3CA Mutations Render Cancer Cells Sensitive to EGCG and BPTES

Epigallocatechin gallate, an active component of green tea extract, hasbeen shown to inhibit glutamine dehydrogenase, whereas BPTES inhibitsglutaminase activity. To test if PIK3CA mutations render cancer cellsensitive to these inhibitors, we treated PIK3CA-/mut, PIK3CA WT/-HCT116and DLD1 colon cancer cells with various doses of EGCG and BPTES. Asshown in FIG. 13, both HCT116 and DLD1 PIK3CA-/mut cells are moresensitive to growth inhibition by EGCG and BPTES in tissue culture.

The data thus demonstrate that oncogenic PIK3CA mutations reprogramglutamine metabolism through the up-regulation of GPT2 in CRCs. Althoughit has been previously shown that WT K-ras regulates glutaminemetabolism in pancreatic cancers by an up-regulation of aminotransferaseGOT112, it is not clear that oncogenic K-ras mutations render cancercells more sensitive to glutamine deprivation. Moreover, both SW480 andLOVO CRC cell lines harbor oncogenic K-ras mutations, but the two celllines were resistant to glutamine deprivation (FIG. 2E). Thus our datasuggest that mutant K-ras is not a key determinant of glutaminedependency in CRCs. In contrast, the data provide compelling evidencethat oncogenic PIK3CA mutations in CRCs render them more sensitive toglutamine deprivation.

Our data also show that CRC cells harboring PIK3CA mutations, but notthose cells with WT PIK3CA, are sensitive to growth inhibition by AOA, acompound that blocks the conversion of glutamate to α-KG. These findingsconstitute a proof-of-principle that targeting glutamine metabolism canbe a useful approach to treating cancers, such as CRCs, harboring PIK3CAmutations. Our results show that targeting glutamine metabolism canafford a specific form of therapy for cancer patients harboring PIK3CAmutations.

This Example demonstrates that GPT2 is the key determinant of glutaminesensitivity in PIK3CA mutant CRC cells. GPT2 is an aminotransaminasethat converts glutamate to α-KG, which is a TCA cycle intermediate.Metabolic profiling shows that amounts of α-KG are significantly higherin the PIK3CA mutant clones than in the WT clones and that the other TCAcycle intermediates are also higher in the mutant clones than in the WTclone. Moreover, α-KG largely rescues PIK3CA mutant cells from celldeath caused by glutamine deprivation, showing that α-KG is a keymetabolite required for PIK3CA-mutant cell growth. Together, these datashow that up-regulation of GPT2 by PIK3CA mutations produces more α-KGfrom glutamine to replenish the TCA, thereby generating more ATP andintermediates for macromolecule synthesis to sustain rapid growth ofPIK3CA mutant tumors. This is consistent with the observation that boththe ATP concentration and the ATP/ADP ratios were higher in the PIK3CAmutant cells than in the WT cells. We also showed that AOA, apan-aminotransferase inhibitor, suppresses xenograft tumor growth ofPIK3CA mutant CRCs.

It is generally believed that AKTs are the key mediators of theoncogenic signaling of PI3Ks. This Example however describes a novelp110α-PDK1-RSK2-ATF4-GPT2 pathway that regulates glutamine metabolism.We demonstrated that blocking this pathway inhibits PIK3CA mutant tumorgrowth in vitro and in vivo, suggesting that this novel signalingpathway also plays a critical role in tumorigenesis driven by PIK3CAmutations.

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes and modifications within the skill of the art areintended to be covered by the appended claims. All references,publications, and patents cited in the present application are hereinincorporated by reference in their entirety.

TABLE S1Gene induced or reduced in PIK3CA mutant cells comparing to PIK3CA WT cellsFold Change (Mut/WT) TAG HCT116 DLD1 Gene AnnotationUp-regulated in the mutant clones TATATATATCCATAGTG 10.00 2.67 KNTC2Kinetochore associated 2 TGACTGAAGCCTTCCAG 9.00 2.00 PHGDHPhosphoglycerate dehydrogenase TGCCTAGACCAAGAAGT 8.89 2.00 AMY2AAmylase, alpha 2A; pancreatic GTGGCCCCGGCCGCACC 8.00 2.22 ALG12Asparagine-linked glycosylation 12 homolog (yeast, alpha-1,6-mannosyltransferase) TGTGGTGGTGTTTTTTG 8.00 2.50 CASC3Cancer susceptibility candidate 3 GCCAGCCAGTGGCAAGC 7.00 4.00 SMTNSmoothelin TTTATTTGGCAAATTTT 6.67 2.00 LBR Lamin B receptorTAAATTCACCAAATAAA 6.67 2.50 TOMM70A Translocase of outer mitochondrialmembrane 70 homolog A (yeast) GGGTGCTTGGTTGTTTC 6.00 2.33 ATP6AP1ATPase, H+ transporting, lysosomal accessory protein 1 TTTAGTTGGTATGTAAA6.00 2.00 FLJ11342 Abhydrolase domain containing 10 GCCACCGTCCTGCTGTC6.00 2.00 MMP9 Matrix metalloproteinase 9 (gelatinaseB, 92 kDa gelatinase, 92 kDa type IV collagenase) CCTTGGTGCCGGTCACA 6.002.00 TRAF4 TNF receptor-associated factor 4 ATGGCCTGTAACAGTTG 5.56 2.00RAP140 Retinoblastoma-associated protein 140 GCTTTCTTATTTTGTTT 5.00 3.50BIRC5 Effector cell protease receptor 1 TATTTTGGAATTTTCCA 5.00 2.20ORC1L Origin recognition complex, subunit 1- like (yeast)TTATGCCTCCATTTTCA 5.00 2.00 SIX5 Sine oculis homeobox homolog 5(Drosophila) ATGTCCAATTTTGTTGT 5.00 2.22 SUCLG2Succinate-CoA ligase, GDP-forming, beta subunit CCAAGGACTCTAGGTCA 5.002.67 SUPT4H1 Suppressor of Ty 4 homolog 1 (S. cerevisiae)TTTTAATTGCTTGTACA 5.00 3.00 TBC1D14 TBC1 domain family, member 14CTTCGACGAATTAAGGA 5.00 3.00 TRA1 Tumor rejection antigen (gp96) 1GGGGGTTGGTTCTTTGG 4.50 6.00 ARL10B ADP-ribosylation factor-like 10BGTGATTCATTTGATGCT 4.50 2.33 C7orf30 Chromosome 7 open reading frame 30AGTTGGACGGACCCCAG 4.50 3.00 LIG1 Ligase I, DNA, ATP-dependentTGCGTCACCGTCCACTC 4.50 3.00 SMARCA4 SWI/SNF related, matrix associated,actin dependent regulator of chromatin, subfamily a, member 4TATAAAATTTAAAAAAA 4.44 2.08 FH Fumarate hydratase GCAAAGAAAAAAAAAAG 4.443.50 LMO4 LIM domain only 4 GAGGGCCGTGTAGCCAT 4.44 2.00 MAPBPIPMitogen activated protein binding protein-interacting proteinGAATGATTTCTCTGCTA 4.44 4.44 ORF1-FL49 Putative nuclear protein ORF1-FL49CAGATTTCTGTATGTTC 4.44 3.00 PRPF4B PRP4 pre mRNA processing factor 4homolog B (yeast) GATCCAAATGTTTGTTG 4.44 3.50 ZWINTZW10 interactor antisense CAGTCAGGCTGGCAGTG 4.33 4.00 SLC26A6Solute carrier family 26, member 6 TGACCGGCGAGCGCGGG 4.25 2.00 TIMM13Translocase of inner mitochondrial membrane 13 homolog (yeast)GTGCTGGTCCCTCCCTT 4.00 2.00 C6orf129 Chromosome 6 open reading frame 129GTATATCATTTCCTCTT 4.00 2.00 CBX3 Chromobox homolog 3 (HP1 gammahomolog, Drosophila) TATGCTTAGTATAAATG 4.00 4.00 CGGBP1CGG triplet repeat binding protein 1 GTGGGTGTCCTGGGGCC 4.00 2.20 COBRA1Cofactor of BRCA1 TGCGCGCCCTGCCGGCG 4.00 2.22 CXXC5 CXXC finger 5TTTGTTAAAACAAAAAA 4.00 11.11 DDIT4 DNA-damage-inducible transcript 4TTCAAGGAACAGGAAAA 4.00 2.22 FLJ11730 Sarcoma antigen NY-SAR-91GTTTCCAAAAAATGGTA 4.00 3.00 GJB2 Gap junction protein, beta 2, 26 kDa(connexin 26) TCAAAAAAAAAAAAAAA 4.00 2.00 HIG1Likely ortholog of mouse hypoxia induced gene 1 GGGGTCCTTCAGCCAGC 4.002.50 KIAA0082 KIAA0082 CCTGTAATCCCAACACT 4.00 2.22 MLL5Myeloid/lymphoid or mixed-lineage leukemia 5 (trithorax homolog,Drosophila) AGCACAAGACTTGTAGT 4.00 3.00 NME2Non-metastatic cells 2, protein (NM23B) expressed in ACTCTGCCAAGCATCCA4.00 2.00 POLDIP2 Polymerase (DNA-directed), delta interacting protein 2CAAACTGCTTTATTTTC 4.00 2.00 PRKAR1A Protein kinase, cAMP-dependent,regulatory, type I, alpha (tissue specific extinguisher 1)TATCGTTGCCTCTGCAC 4.00 3.00 PSMD1 Proteasome (prosome, macropain) 26Ssubunit, non-ATPase, 1 AGAGTTTGAGGCTTCAG 4.00 4.44 SRPRBSignal recognition particle receptor, B subunit TGTGAGTTATTATCACT 4.003.33 TRIT1 TRNA isopentenyltransferase 1 GTCATATTTCCTGAGTA 3.50 3.50DPCD Deleted in a mouse model of primary ciliary dyskinesiaTCAGATAGGACAACACT 3.50 2.00 MAPKAPK3 Mitogen activated protein kinaseactivated protein kinase 3 ATGTTCAATTTTATCTT 3.33 3.50 ABCB10ATP binding cassette, sub-family B (MDR/TAP), member 10CCTGGCTAATTTTTGTA 3.33 3.00 AMID Apoptosis-inducing factor (AIF)-likemitochondrion-associated inducer of death GTGAATCTATCTCTCCG 3.33 2.00BDH 3-hydroxybutyrate dehydrogenase (heart, mitochondrial)CAGTGTATATATTGAGA 3.33 2.56 BRP44L Brain protein 44-likeGGCCTATGAGCGGTCTA 3.33 2.33 C1orf35 Chromosome 1 open reading frame 35TATATAAGTACTGACCA 3.33 2.22 CTNND1Catenin (cadherin-associated protein), delta 1 GGTGTCTGTGGGTTATT 3.332.00 DGAT2 Diacylglycerol O-acyltransferase homolog 2 (mouse)ACTTGATAAATTAAGTA 3.33 2.00 EIF4G2Eukaryotic translation initiation factor 4 gamma, 2 ACATAAATAAAAAAATA3.33 2.22 FLJ12949 Hypothetical protein FLJ12949 ACTTACCTGTAATGGGA 3.332.22 FLJ20323 Hypothetical protein FLJ20323 CGCTGAATGATGTCACG 3.33 4.00GNAS GNAS complex locus CCTGTCTAGCTCATACA 3.33 3.33 IMP-2IGF-II mRNA-binding protein 2 TAACCTCAGGTATCTTC 3.33 2.22 KIAA0870KIAA0870 protein CTCTGTTACCTGGTGAA 3.33 3.33 NKAPNF-kappaB activating protein CTCTCCTGCTCAAGGCA 3.33 2.22 PRDM4PR domain containing 4 TGGATGTACTTATGACC 3.33 2.00 PSMD14Proteasome (prosome, macropain) 26S subunit, non-ATPase, 14TGCACGTTCTCTGTTTA 3.33 2.00 RPL32 Ribosomal protein L32GAAAACTGTTTATTTTT 3.33 2.22 RYK RYK receptor-like tyrosine kinaseAATTATGACTTCTCATT 3.33 3.00 SARA1 SAR1a gene homolog 1 (S. cerevisiae)CTCCAGGAGGATGAGCT 3.33 2.00 SYAP1 Synapse associated protein 1, SAP47homolog (Drosophila) CAGTCGGTCAAGAGGAG 3.00 2.22 APAF1Apoptotic protease activating factor CATTTCAGAGACTTTAA 3.00 2.67 BAG3BCL2-associated athanogene 3 AACCTCGAGTTCTGACT 3.00 2.22 BAT4HLA-B associated transcript 4 TAACAGTTGTGTCATAA 3.00 2.67 CANX CalnexinGCCCAGGGCCGCTGGGA 3.00 2.00 COPE Coatomer protein complex, subunitepsilon AAAGAAACCCTGCGGAT 3.00 2.22 DHRS4Dehydrogenase/reductase (SDR family) member 4 like 2 CACCTAGCATAGTGCTT3.00 2.00 DHX40 DEAH (Asp-Glu-Ala-His) box polypeptide 40AGGCTAAAAGCAAAGTC 3.00 3.00 DLNB14 Similar to DLNB14 GTGATGGGCTCCCTCCC3.00 2.00 EVPL Envoplakin GCTACACACACCCTTGC 3.00 2.00 FAM32AFamily with sequence similarity 32, member A AATAAAGTCATTACTAG 3.00 2.00FLJ11171 Hypothetical protein FLJ11171 TAATCAGGAGAAAGGGA 3.00 3.33FLJ20014 Hypothetical protein FLJ20014 TGTGACACTGATTCTTT 3.00 2.00FLJ23825 Hypothetical protein FLJ23825 TGAATGATTTTCTCAAA 3.00 5.00 GBASGlioblastoma amplified sequence GCCCCAGCGAGGGGCTG 3.00 3.50 GGA1Golgi associated, gamma adaptin ear containing, ARF binding protein 1TTAAATAAAACCATTTT 3.00 2.33 GGA3 Golgi associated, gamma adaptin earcontaining, ARF binding protein 3 GAAATAAAATTACTTAT 3.00 2.25 gm117Chromosome 1 open reading frame 52 ATCTGTGAAATAAAGCC 3.00 6.00 GPT2Glutamic pyruvate transaminase (alanine aminotransferase) 2CTATGCATCAGACTGGC 3.00 3.00 HCA66 Chromosome 17 open reading frame 40TGGCTTAAATGATTTTT 3.00 2.00 HIG2 Hypoxia-inducible protein 2CTGCTTCAGCAGTGACG 3.00 3.33 HIRA HIR histone cell cycle regulationdefective homolog A (S. cerevisiae) TCTACTCAGCATTTGAT 3.00 2.00 HMMRHyaluronan-mediated motility receptor (RHAMM) AGACCAGGCAAGAAGGT 3.002.22 HSPC159 HSPC159 protein TCTGCCTATGCACTGAA 3.00 3.33 LAMB2Laminin, beta 2 (laminin S) GGGGTCCCAAACAGTCA 3.00 2.22 LOC93622Hypothetical protein BC006130 TGAGTGGTCACTTTATT 3.00 2.00 MAP1LC3BMicrotubule-associated protein 1 light chain 3 beta CCCCAGACCGGCCCACC3.00 2.22 MAP3K7IP1 Mitogen activated protein kinase kinasekinase 7 interacting protein 1 TCCTTTTTTGTGGACTT 3.00 2.00 MARCH-VIMembrane associated ring finger (C3HC4) 6 TTGCCGCTGCTGTTTCT 3.00 2.00MGC3731 Hypothetical protein MGC3731 GTGGGCCTTTGAGGTTC 3.00 3.33 MSRAMethionine sulfoxide reductase A ACATCTTGCTTATAAAT 3.00 2.00 NEK2NIMA (never in mitosis gene a) related kinase 2 AACAAGTCTTTCTAATG 3.004.00 NOLA1 Nucleolar protein family A, member 1(H/ACA small nucleolar RNPs) GTACCCGTACAGCGTTG 3.00 2.00 PDXPPyridoxal (pyridoxine, vitamin B6) phosphatase ACCTCCACCAAAGCCCA 3.003.00 POLR2D Polymerase (RNA) II (DNA directed) polypeptide DTGTCTGGTTGTTTGAAA 3.00 5.00 RPS24 Ribosomal protein S24GCAGGCGGCTCTGGCTT 3.00 2.00 RPS3 Ribosomal protein S3 CAATAAAACAAACTCTA3.00 2.14 SARA2 SAR1a gene homolog 2 (S. cerevisiae) GTATCTTAATAAAGAAT3.00 2.00 SYNCRIP Synaptotagmin binding, cytoplasmicRNA interacting protein TGAACACCCGTGTCTGG 3.00 4.44 UCK1Uridine-cytidine kinase 1 ATTTATCCATAAAGGAG 3.00 2.22 ZFP161Zinc finger protein 161 homolog (mouse) ACATTCTTGTTTTTAAT 3.00 4.00 ZFRZinc finger RNA binding protein GGACTGGCCCAGGCACA 2.75 2.00 NUDCNuclear distribution gene C homolog (A. nidulans) GATAGAGGGACTGAGGG 2.673.33 FLJ20257 Hypothetical protein FLJ20257 GGGGGCAGGTCCCCCAG 2.60 7.00CBX6 Chromobox homolog 6 GGGTTCCCCGGCAGGGG 2.50 2.00 CENTD2Centaurin, delta 2 CCCTCGCATTGCTTCCC 2.50 2.00 CHTF18CTF18, chromosome transmissionfidelity factor 18 homolog (S. cerevisiae) TTATTTTCCTGTGTCAT 2.50 3.20DDX18 DEAD (Asp-Glu-Ala-Asp) box polypeptide 18 GGCAGGCGGGTGGGGGG 2.502.00 ERF Ets2 repressor factor GGGCAAGCCAGGGCCCA 2.50 2.40 ESRRAEstrogen-related receptor alpha GGCCCTGGTGTTTGCAC 2.50 3.00 GAKCyclin G associated kinase GTGGCGGGAGCCTGTTG 2.50 2.00 GTF2F1General transcription factor IIF, polypeptide 1, 74 kDaGTGCCATATTTAGCTAC 2.50 2.00 IDH2 Isocitrate dehydrogenase 2 (NADP+),mitochondrial GCCCCTGCGCAAGGATG 2.50 2.00 KLHL7Kelch-like 7 (Drosophila) GGAAGAGGGTGAGCTGA 2.50 3.00 LASS5LAG1 longevity assurance homolog 5 (S. cerevisiae) ACCATAATGTGTTTAAA2.50 2.00 NIF3L1BP1 Ngg1 interacting factor 3 like 1 binding protein 1ACCCAATTTGTGTTATT 2.50 2.22 PAN3 PABP1-dependent poly A-specificribonuclease subunit PAN3 CGCGCACCCGCCGACCC 2.50 2.00 PNPLA2Patatin-like phospholipase domain containing 2 TCCTGAAATAAATATTG 2.505.56 RAB6A RAB6A, member RAS oncogene family GAGTTACTGAAGGTCTC 2.50 3.00RPL36 Ribosomal protein L36 GCGTGTGCTCGCCCACT 2.50 4.00 RPL4Ribosomal protein L4 AAGAAGCAAGACGAAAA 2.50 2.00 RPS15ARibosomal protein S15a AGAGACAAGTCTCTTAG 2.50 2.00 RRBP1Ribosome binding protein 1 homolog 180 kDa (dog) TGATGTCCACCAGTGGA 2.507.00 SNX6 Sorting nexin 6 TCCCTGGGCAGCTTCAG 2.50 2.00 SOX4SRY (sex determining region Y)-box 4 TAAATTACCAGTAAAGT 2.50 3.00 SP3Sp3 transcription factor CCTGACGCTCCAGCGCC 2.50 4.00 YTHDF1YTH domain family, member 1 GGCCTCTCAAGGCTGGC 2.33 4.00 CYHR1Cysteine and histidine rich 1 GACTCAGGGATTTGTTG 2.33 2.00 GTPBP2GTP binding protein 2 GGGGACGGGAGGAGGGG 2.33 2.67 HSPBP1Hsp70-interacting protein GTAAAAAAGCCTGAAAC 2.33 2.00 LOC146439Hypothetical LOC146439 TACTAAAAAAGGAGAAA 2.33 3.67 NDUFS2NADH dehydrogenase (ubiquinone) Fe- S protein 2, 49 kDa (NADH-coenzyme Qreductase) TGCCTTGAAAGGGGGCA 2.33 2.00 NOC4 Neighbor of COX4CCAGGAACAATGTCTCC 2.33 2.50 SNX5 Sorting nexin 5 TTTGCTGAACACCTTGT 2.223.00 ABCC5 ATP binding cassette, sub-family C (CFTR/MRP), member 5GTTTGCGGAGGTTAGAT 2.22 2.00 ARFGEF1 ADP-ribosylation factor guaninenucleotide-exchange factor 1(brefeldin A-inhibited) TTGAACTGGCCTCTTTT2.22 2.00 ARIH2 Ariadne homolog 2 (Drosophila) TAGCAAAGATTTTCAAA 2.222.00 ARNT Aryl hydrocarbon receptor nuclear translocatorTTTCAATACCTACAAAC 2.22 3.33 ASXL2 Additional sex combs like 2(Drosophila) TGATTTCTGTACATAAG 2.22 2.00 ATP5A1 ATP synthase, H+transporting, mitochondrial F1 complex, alphasubunit, isoform 1, cardiac muscle TATTTTCTTTGTAAAGT 2.22 3.33 C14orf106Chromosome 14 open reading frame 106 TACCGGGAATACCGGGA 2.22 2.22C14orf130 Chromosome 14 open reading frame 130 GGCTAGTACTTGGGGTT 2.223.00 C15orf19 RNA pseudouridylate synthase domain containing 2GTGGTGGGTGCCTGTAA 2.22 2.22 C21orf4 Transmembrane protein 50BTACTTTCTCCTTTCTGG 2.22 4.44 C5orf3 Chromosome 5 open reading frame 3TGAGGAGGTTGCGCGCT 2.22 2.22 C9orf114 Chromosome 9 open reading frame 114TGCCACCACGCCCAGCT 2.22 2.22 CA6 Carbonic anhydrase VI TAAAATCAAAATATAAG2.22 6.00 CANX Calnexin CACTACGGGAGCTAGGG 2.22 2.22 CARM1Coactivator-associated arginine methyltransferase 1 GTAATTATTGGAAAGTA2.22 3.00 CDA08 T-cell immunomodulatory protein TTAAATCGTGACAGAAT 2.222.22 CGI-143 BolA-like 1 (E. coli) GCTCGTGGTCAAAAAAG 2.22 2.00 CIRBPCold inducible RNA binding protein AGCAGCAGAGTCGAGTG 2.22 2.50 CLUClusterin (complement lysis inhibitor,SP-40, 40, sulfated glycoprotein 2,testosterone-repressed prostate message 2, apolipoprotein J)AGGGACTTGTGTGACCT 2.22 2.00 CPT1B Carnitine palmitoyltransferase 1B(muscle) ATTCAAATTCTTCAAAG 2.22 2.22 D8S2298E Reproduction 8TTCCCTCGTGATCCCAA 2.22 2.00 DARS Aspartyl-tRNA synthetaseTTTGTTAAAAAAAAAAA 2.22 5.00 DDIT4 DNA-damage-inducible transcript 4TGGGCAATATCCCAGTT 2.22 6.00 DKFZP564K0822EGFR-coamplified and overexpressed protein TAGTTGTTTAGTTATAA 2.22 5.56DNAJC10 DnaJ (Hsp40) homolog, subfamily C, member 10 GTTTTTATTCACTTGAA2.22 2.00 E2F4 E2F transcription factor 4, p107/p130- bindingTTCAACTTTTTATTGTG 2.22 2.00 ECT2 Epithelial cell transforming sequence 2oncogene CAATAAAACTGATTGTC 2.22 2.00 ELP3Elongation protein 3 homolog (S. cerevisiae) GGAGAGTAACATCACAG 2.22 2.00FLJ10707 Hypothetical protein FLJ10707 GCATAATTACTTGGTAG 2.22 2.22FLJ14888 WD repeat domain 73 GCACAAGAGAAACCAGC 2.22 2.00 FLJ20259FLJ20259 protein TATTCCCCACCTGTGTT 2.22 2.00 FOXP4 Forkhead box P4GCCTTCCGTGTCCCCAC 2.22 5.56 GAPD Glyceraldehyde-3-phosphatedehydrogenase TGCTGAGGAAGCACGTG 2.22 3.00 GPT2Glutamic pyruvate transaminase (alanine aminotransferase) 2CCCAAAGACATCCAGTT 2.22 2.22 H3F3B H3 histone, family 3B (H3.3B)TTCTAAACTGTTTTTTC 2.22 3.33 HH114 Hypothetical protein HH114AGGCCGTCCCCGAAGGC 2.22 2.22 HNRPABHeterogeneous nuclear ribonucleoprotein A/B CCACCTCCCATACCACC 2.22 2.22HSPD1 Heat shock 60 kDa protein 1 (chaperonin) GACAAATACATCCACAA 2.222.22 IL17RC Interleukin 17 receptor C GGCAAGTGCAAGGTGTA 2.22 2.22 IRF7Interferon regulatory factor 7 TTTCAAAGATACAGTAT 2.22 3.50 KIAA0073Peptidylprolyl isomerase domain and WD repeat containing 1GGTCCAGCATCAGGCCT 2.22 2.00 KIAA0376 KIAA0376 protein CACCATCAAAAAAAAAA2.22 7.00 KIAA1185 Leucine rich repeat containing 47 TACTGCATTGTTACTTT2.22 2.00 KIAA1333 KIAA1333 TTGTTGAAGCAAATGAA 2.22 2.22 KIF4AKinesin family member 4A CCCTTCTGCCATCTTCT 2.22 2.00 LARPLa ribonucleoprotein domain family, member 1 TTTGTGAATATTTTATA 2.22 2.22LIN7C Lin-7 homolog C (C. elegans) GGGTGCAAAAAAAAAAT 2.22 2.00 MAPKAP1Mitogen-activated protein kinase associated protein 1 TATACTTTGATTTCAAC2.22 2.00 MGC14817 Hypothetical protein MGC14817 AAGGCAAAGCTCTTGTA 2.222.00 MGC23908 Basic transcription factor 3-like 4 GAGTAACTTAAAAATAC 2.222.22 MGC29816 CHMP family, member 7 CCGCTGCACTCCAGCCT 2.22 2.00 MRP63Mitochondrial ribosomal protein 63 CCTGTCTGATAATCTTG 2.22 2.00 MYCBPC-myc binding protein TACAAAACCATTTTTTT 2.22 2.00 NCL NucleolinGTTACAATCATTGCTGA 2.22 3.00 NFKB1Nuclear factor of kappa light polypeptidegene enhancer in B-cells 1 (p105) ATCTCTGGGCACACAGC 2.22 4.44 NOB1PNin one binding protein GAGGAATTTGTAACGAT 2.22 3.00 NOC4Neighbor of COX4 CTTCACTCGTGGGCCAG 2.22 3.33 NOP5/NOP58Nucleolar protein NOP5/NOP58 CATTGGTAGAATCGTGT 2.22 4.44 NUCKSNuclear ubiquitous casein kinase and cyclin-dependent kinase substrateTCCTTTTGCTTACTGTT 2.22 2.22 OSBP2 Oxysterol binding protein 2AAGGTAACTTGGGTTTT 2.22 2.00 PANK3 Solute carrier family 2 (facilitatedglucose transporter), member 3 pseudogene 1 ACAAACAGAAAAATTCA 2.22 3.33PCBP2 Poly(rC) binding protein 2 CGGGGACGAGGACCTGG 2.22 3.33 PR48Protein phosphatase 2 (formerly 2A), regulatory subunit B″, betaACGTCTCTATTGTACAA 2.22 4.00 PRPF4 PRP4 pre mRNA processing factor 4homolog (yeast) TATTATTAAAGAGGATT 2.22 2.40 RAB20RAB20, member RAS oncogene family TCAGACTTTGAGCTGAT 2.22 2.00 RAMPDenticleless homolog (Drosophila) TTAATAAACAAAGTAAC 2.22 2.67 RCBTB1Regulator of chromosome condensation (RCC1) and BTB (POZ) domaincontaining protein 1 TATATAGTGAGATGTCT 2.22 2.22 RCOR3REST corepressor 3 CACCTATCAATGTGTTT 2.22 4.00 ROCK2Rho-associated, coiled-coil containing protein kinase 2TTGTAGCTCAATACAAT 2.22 2.00 SAFB2 Scaffold attachment factor B2TCACACTGGCTATCAAA 2.22 2.22 SIL TAL1 (SCL) interrupting locusACTTTATTTTTGTTGGG 2.22 3.33 SLC25A17Solute carrier family 25 (mitochondrialcarrier; peroxisomal membrane protein, 34 kDa), member 17GACATCACAAGACCATC 2.22 2.00 SLC7A6Solute carrier family 7 (cationic amino acid transporter, y+system), member 6 GTAAACACCATTTCCCA 2.22 2.22 STATIP1Signal transducer and activator of transcription 3 interacting protein 1TCTGCAAGCAGTTCTTC 2.22 2.00 T1 IKK2 binding protein TTTATCATCTTTACTTT2.22 2.00 TCP1 T-complex 1 TAAAATACTCCACAATA 2.22 2.00 TOM1L2Target of myb1-like 2 (chicken) AAATTGAATTTCCCGAT 2.22 2.00 TTKTTK protein kinase GTGTGGTCACTGTCAAA 2.22 2.50 TXNDC7Protein disulfide isomerase family A, member 6 TTGGTTTTAATAGTGTC 2.222.00 UGDH UDP-glucose dehydrogenase TGGCTAGATTTATGCTA 2.22 2.50 UMP-CMPKCytidylate kinase GGTAGTTTTAAATAAAT 2.22 4.00 XBP1X-box binding protein 1 TTTGTCGGTCCGGGCTT 2.22 2.22 ZDHHC6Zinc finger, DHHC-type containing 6 TGAGATACAAGGCTACA 2.22 3.00 ZRF1Zuotin related factor 1 GGCGTCCTGGCCGCAGC 2.20 2.17 MRPL41Mitochondrial ribosomal protein L41 GAGTTAGGCACTTCCTG 2.00 2.50 ACBD3Acyl Coenzyme A binding domain containing 3 TGCACTTGACCTGACAG 2.00 2.00ADIPOR2 Adiponectin receptor 2 CCCAGCAAGAGCCTTGC 2.00 2.00 ARF3ADP-ribosylation factor 3 CCTAGGACCTGGGGCCC 2.00 2.00 ARPC4Actin related protein 2/3 complex, subunit 4, 20 kDa ATATTTTAAATGTTAAG2.00 2.00 B3GNT1 UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 1 TACTTGTGTTTATAAAA 2.00 4.00 BAZ1BBromodomain adjacent to zinc finger domain, 1B AAGAACTAAAAAAAAAA 2.002.33 BET1L Blocked early in transport 1 homolog (S. cerevisiae) likeACCAGAGAGCATTTAGG 2.00 2.00 C10orf61 Chromosome 10 open reading frame 61CAATCAGAATCTCACTG 2.00 2.00 C14orf2 Chromosome 14 open reading frame 2ATTAAAGAATGCTGTCT 2.00 2.22 C1QTNF6C1q and tumor necrosis factor related protein 6 ATTCTGCTTTCTGTTAG 2.002.22 C20orf11 Chromosome 20 open reading frame 11 ATGTACAGGTTTGTAGC 2.002.50 C20orf129  Chromosome 20 open reading frame 129 CTGGCAGGCCACAGCCC2.00 2.00 C20orf29 Chromosome 20 open reading frame 29 TAACTGTCTTAAAAAAA2.00 2.29 C6orf115 Chromosome 6 open reading frame 115 TGATGGGTGGGGTGCCT2.00 2.00 CCT7 Chaperonin containing TCP1, subunit 7 (eta)TTGCTATGATGGACAGG 2.00 2.22 CDC42SE1 CDC42 small effector 1GGCCCGGCTTTCCTGGA 2.00 2.00 CHCHD5 Coiled-coil-helix-coiled-coil-helixdomain containing 5 TTAAGAGAAGGGAGTGT 2.00 4.44 CIAPIN1Cytokine induced apoptosis inhibitor 1 ATTATCACATTCTGCCA 2.00 2.00CSNK1A1 Casein kinase 1, alpha 1 AACGTTCTTGTCTGTGT 2.00 2.22 CTBP1C-terminal binding protein 1 GATGGGCTGCCTCCAGG 2.00 2.00 CTNNB1Catenin (cadherin-associated protein), beta 1, 88 kDa TGGGGAGCTCGGCTGCA2.00 3.00 CXorf37 Chromosome X open reading frame 37 GAAAAAATAAAGCCATT2.00 2.00 DHODH Dihydroorotate dehydrogenase CACTGCAAGGCTGTGAC 2.00 2.00DKFZp761A132 FLYWCH-type zinc finger 1 GCTGGAGCTAGAATTTG 2.00 2.00 DLDDihydrolipoamide dehydrogenase (E3 component of pyruvate dehydrogenasecomplex, 2-oxo-glutarate complex, branched chain keto acid dehydrogenasecomplex) TGTCTGCCTGACCCGTA 2.00 2.00 DPP9 Dipeptidylpeptidase 9AGGTGTCTTTGCAGAAC 2.00 3.50 DTX4 Deltex 4 homolog (Drosophila)AAGATCCTACGAGAGAT 2.00 3.00 F25965 Protein F25965 CTAATGGGGTACACCAT 2.002.00 FBXW2 F-box and WD-40 domain protein 2 ATGAAAGGTGTCAATAA 2.00 2.00FGFR1OP FGFR1 oncogene partner AGTCAAGCCCCCTCCCC 2.00 2.22 FHL3Four and a half LIM domains 3 TTTCTCATACCCAGATA 2.00 2.00 FLJ10514Aspartyl-tRNA synthetase 2 (mitochondrial) TTCTTCATTATATCTTC 2.00 2.00FLJ11305 Hypothetical protein FLJ11305 AAGAAATTCTTCTGTCT 2.00 5.00FLJ11506 Hypothetical protein FLJ11506 TTACTCTTAGTAAATAA 2.00 3.33FLJ13149 Hypothetical protein FLJ13149 GACATTTGTCCTCGGGC 2.00 3.00FLJ14154 Hypothetical protein FLJ14154 GTGGCAGCCGGAGGTGC 2.00 2.00FLJ14640 Hypothetical protein FLJ14640 TCAATATCACTGTTTTT 2.00 2.67FLJ20457 Hypothetical protein FLJ20457 ATGACCTGAAGTTCACC 2.00 2.00 FVT1Follicular lymphoma variant translocation 1 AACCTCTGTATTGCTTT 2.00 2.22GCLM Glutamate cysteine ligase, modifier subunit GATCTGTTCCTCTGTGC 2.002.00 GLE1L GLE1 RNA export mediator-like (yeast) GTGTCCTCCTCCTCCTC 2.004.00 GLG1 Golgi apparatus protein 1 CATTGTCTTCACGAAGA 2.00 3.00 GLRX2Glutaredoxin 2 ACTTATGTTTATTACTA 2.00 2.50 GMNNGeminin, DNA replication inhibitor AGTTTGTTAAATAGCTT 2.00 2.22 GTF2A1General transcription factor IIA, 1, 19/37 kDa TTGCCCAGGCTGGTCTT 2.002.00 HIF3A Hypoxia inducible factor 3, alpha subunit CCCTCCTGCTCCCCCCA2.00 3.00 HIP1R Huntingtin interacting protein-1-relatedCGTGGGTGGGGAGGGAG 2.00 2.50 HMOX1 Heme oxygenase (decycling) 1AATATTAGAGAAGGAAT 2.00 2.00 HRSP12 Heat-responsive protein 12CCTGGAGCAATGAGGGT 2.00 2.33 HSPC171 HSPC171 protein GCTTGCTGGCCAGGATA2.00 3.00 HTF9C HpaII tiny fragments locus 9C TTAATAAACAGGAACAC 2.002.22 INPP4B Inositol polyphosphate-4-phosphatase, type II, 105 kDaTAGCAATCAGATTTTCC 2.00 2.00 IRF2 Interferon regulatory factor 2CGTGCCTGCTGGGGAGG 2.00 2.00 KIAA0258 KIAA0258 AGCACCAGAACAGATGA 2.004.00 KIAA0690 KIAA0690 CCCCAAGACACAGGGAC 2.00 2.00 KIAA0963 KIAA0963GGGGCAGTGAGACCAGG 2.00 2.22 KIAA1285 KIAA1285 protein CTAGAAAGAGCTCAGTG2.00 3.33 KIAA1967 KIAA1967 CAAATGAATTTTTGGTG 2.00 3.00 LACTB2Lactamase, beta 2 GGTTGAGTGTGGCCACC 2.00 3.00 LOC127262Hypothetical protein LOC127262 GCGAAACCCCGTCTCTA 2.00 2.00 LOC284912Hypothetical gene supported by BC001801 AGTCTTCTGACTCTGTT 2.00 5.56MAP4K5 Mitogen activated protein kinase kinase kinase kinase 5TTTGGAATGTTAAAAAA 2.00 2.33 MATR3 Matrin 3 CGGTCCCATTGTGAAAT 2.00 3.33MGC11134 TRNA splicing 2′ phosphotransferase 1 GTTGTAGACTTTCACCT 2.002.00 MGC24665 Hypothetical protein MGC24665 TTACTTTTTCCTTTGCT 2.00 2.22MGC26717 Hypothetical protein MGC26717 CGAGGCTGTAGGAAGAG 2.00 2.00MGC3265 Hypothetical protein MGC3265 GGTGAACTTTATGAGTG 2.00 3.33 MICBMHC class I polypeptide related sequence B CCTGACCTCAACCCCGC 2.00 2.00MKLN1 Muskelin 1, intracellular mediator containing kelch motifsTTTGTATAGAAAAAATG 2.00 2.00 MN7 D15F37 gene GACCAGCCTTCAGATGG 2.00 3.00MRPL45 Mitochondrial ribosomal protein L45 TAATTTGGAAGGGCTCA 2.00 4.00MTIF2 Mitochondrial translational initiation factor 2 GCCCCGTGAGGGTTTTG2.00 6.00 MYCBP C-myc binding protein AATGCTTTACCATTCAA 2.00 2.00 NEK7NIMA (never in mitosis gene a)-related kinase 7 CAGACTGCCCTGCTGGG 2.003.00 NFIX Nuclear factor I/X (CCAAT-binding transcription factor)GGAGGTGTGGTTTATTG 2.00 3.00 NY-REN-41 NY-REN-41 antigenGTTGCAGATAAACTGAT 2.00 2.00 OGT O-linked N-acetylglucosamine (GlcNAc)transferase (UDP-N- acetylglucosamine:polypeptide-N-acetylglucosaminyl transferase) ACAATGAAGCAGATATG 2.00 2.22 PCGF1Polycomb group ring finger 1 TTCTGAAGACAAATTTT 2.00 2.22 PDIRProtein disulfide isomerase family A, member 5 ATAAATTAGACCCAGTC 2.002.00 PERP PERP, TP53 apoptosis effector TATATACATTTGGAAAT 2.00 2.00PMPCB Peptidase (mitochondrial processing) beta TCAGAAGTTCCTAATTC 2.002.00 POP5 Processing of precursor 5, ribonucleaseP/MRP subunit (S. cerevisiae) TATATTTTCTATTAGTT 2.00 2.00 PTBP2Polypyrimidine tract binding protein 2 CTTGTATACATATTTAA 2.00 5.00 RHPN2Rhophilin, Rho GTPase binding protein 2 TCGGAGCTGCTGGAGCC 2.00 2.22 RIN1Ras and Rab interactor 1 CAACAGTTGTCCTAAGG 2.00 2.00 RNP24Coated vesicle membrane protein GTTGATGGTGGGGTCCC 2.00 2.22 RPL18Ribosomal protein L18 CGCTACTTAATGGAAGA 2.00 2.00 RPL5Ribosomal protein L5 GTGCCCACAGGGAGCAC 2.00 2.22 RPL8Ribosomal protein L8 ATGTGAGGGAGATGAGA 2.00 3.00 SECP43TRNA selenocysteine associated protein TTTTGAAGATTGTTTAA 2.00 2.22 SENP7SUMO1/sentrin specific protease 7 AAGGAGCGGGAACGCCG 2.00 2.22 SFRS16Splicing factor, arginine/serine-rich 16(suppressor-of-white-apricot homolog, Drosophila) GCTCTGCCGTCCTGCCT 2.002.22 SLC2A4RG SLC2A4 regulator TAGCAGCAATGCAGATT 2.00 7.00 SLC39A8Solute carrier family 39 (zinc transporter), member 8 GCCTCTTTCCTTGGACA2.00 2.22 SLU7 Step II splicing factor SLU7 CAGGATTTAATATTTTC 2.00 2.00SMBP SM-11044 binding protein GGTAGCTCAGGGGAGGA 2.00 3.33 SPINL SpinsterGTCTACCTGATCCGGGT 2.00 4.00 SPINT2Serine protease inhibitor, Kunitz type, 2 CGGCTTTTCTGCATCAA 2.00 2.00SPTBN1 Spectrin, beta, non-erythrocytic 1 TGCACACGTGCCCAGGC  2.00 2.22SSB1 SPRY domain containing SOCS box protein SSB-1 TTGAAATATATGTGTTG2.00 2.00 SYNCOILIN Syncoilin, intermediate filament 1 CAGAGAATATATATTGT2.00 2.00 TNPO1 Transportin 1 GGCATCAGGGGCTGGCC 2.00 3.00 TSK TsukushiGAGACCTTCTTCTACCG 2.00 2.00 U2AF1L3 U2(RNU2) small nuclear RNA auxiliaryfactor 1-like 3 GGAGTAATAATGGGTCA 2.00 2.50 UBE2D3Threonine synthase, chloroplast TGTGTTAAGAAACACTG 2.00 2.22 UBE2NUbiquitin conjugating enzyme E2N (UBC13 homolog, yeast)GCTGAGAATATGACGGC 2.00 2.00 UBQLN4 Ubiquilin 4 GTGAAACTAGTGATGAA 2.002.33 UCHL3 Ubiquitin carboxyl-terminal esterase L3(ubiquitin thiolesterase) GAGAAACCCTGTCTCTA 2.00 3.50 VDRVitamin D (1,25-dihydroxyvitamin D3) receptor CTGTTATAGGATCTACA 2.006.00 YME1L1 YME1-like 1 (S. cerevisiae) GCCACACTGTCAGTGAG 2.00 4.00ZCWCC2 MORC family CW-type zinc finger 4 CAAAATACTGCAGATTT 2.00 3.00ZNF161 Zinc finger protein 161 CACATCCCCACCTCGGG 2.00 2.22 ZNF503Zinc finger protein 503 Down-regulated in the mutant clonesACTACCTCCCCCAGGAG 0.10 0.30 MOV10 Mov 10, Moloney leukemia virus 10,homolog (mouse) ATCCATAGTGAAATTGC 0.10 0.25 TAF15TAF15 RNA polymerase II, TATA boxbinding protein (TBP)-associated factor TCAGATCCGTCGATCCC 0.11 0.45RRAGA Ras-related GTP binding A TGTTGATTTTATTTGAC 0.13 0.44 AGRN AgrinGTTCCAGTGAGGCCAAG 0.14 0.45 FKBP5 FK506 binding protein 5CAGAACCTCAACGACCG 0.14 0.30 KRT19 Keratin 19 GGATGGCAATGTCCACA 0.14 0.33LAMR1 Ribosomal protein SA AAGTGATTCTGTTGACA 0.14 0.40 SF1Splicing factor 1 TTCCCAAAGGCCAGCGG 0.15 0.45 ARFRP1ADP-ribosylation factor related protein 1 ACTTCACAAAGACCCTA 0.15 0.33FLJ22405 Hypothetical protein FLJ22405 TAAGAACTAAGAGTTCT 0.17 0.23 PCNPPEST-containing nuclear protein CAGGACAGTTTTTCAAC 0.17 0.23 RAB2RAB2, member RAS oncogene family CTGTGCCCAGTTCAATA 0.17 0.20 RPL30Ribosomal protein L30 TTGTTTAATTTCTTTTT 0.18 0.50 CAPZA2Capping protein (actin filament) muscle Z-line, alpha 2TAAATACAGTATGCTCT 0.18 0.50 CPT1ACarnitine palmitoyltransferase 1A (liver) GTTTCAGCACTAGCCAA 0.18 0.45MAT2A Methionine adenosyltransferase II, alpha ATTAAGACAATAAAGTA 0.180.50 STRN3 Striatin, calmodulin binding protein 3 CTGCTTCCAGAGCCCTC 0.180.50 ZW10 ZW10 homolog, centromere/kinetochore protein (Drosophila)CCACAATCCTATGCTCT 0.20 0.25 AK2 Adenylate kinase 2 CTGTGCCAATGGCTGGC0.20 0.18 AP2B1 Adaptor related protein complex 2, beta 1 subunitTAACACTGACTTTATCC 0.20 0.30 BTBD7 BTB (POZ) domain containing 7ATTTGAGATGTAGAAGC 0.20 0.27 CDC45L CDC45 cell division cycle 45-like (S.cerevisiae) TTGGCCAGGATGGTCTT 0.20 0.33 CEBPZCCAAT/enhancer binding protein zeta TTTAATTCAAAGAAGAG 0.20 0.20 DDX46DEAD (Asp-Glu-Ala-Asp) box polypeptide 46 CAGGATCCAGAAGTTAT 0.20 0.43FAM10A5 Family with sequence similarity 10, member A5 TGCTAATTGTAACCACA0.20 0.45 IL6ST Interleukin 6 signal transducer (gp130,oncostatin M receptor) GGCAAGAGACAATTTGG 0.20 0.45 LOC143458Hypothetical protein LOC143458 GAAATCCGCACTTTCCT 0.20 0.30 MAN2B1Mannosidase, alpha, class 2B, member 1 CCCCAAGACCCCAGGGC 0.20 0.33 NARFLNuclear prelamin A recognition factor like GAACTGGATTTGGATTT 0.20 0.50NSDHL NAD(P) dependent steroid dehydrogenase-like CAAGACGGGGGTTAGTG 0.200.50 RALGDS Ral guanine nucleotide dissociation stimulatorTTCATATAGTCAATGTA 0.20 0.50 RDX Radixin AACTTGGGCTTTTCTGG 0.20 0.45 RHOARas homolog gene family, member A GTTAATCTGGAACTTAC 0.20 0.50 RRN3RRN3 RNA polymerase I transcription factor homolog (yeast)GCCAATGTGGGCGGCTT 0.20 0.45 SLC37A4Solute carrier family 37 (glycerol-6- phosphate transporter), member 4CAATTTAAAGTAACTTA 0.21 0.25 EREG Epiregulin TACTGTGATGTCTGATG 0.22 0.33C11orf15  Chromosome 11 open reading frame 15 GTGAAGCTGATGCAGCG 0.220.50 CLPTM1 Cleft lip and palate associated transmembrane protein 1AAATGTAATTTACTTGG 0.22 0.20 EPLINEpithelial protein lost in neoplasm beta CCCCCACCTAAGTCACA 0.22 0.29PLP2 Proteolipid protein 2 (colonic epithelium-enriched)AATTAACTCCGTTAAAA 0.23 0.33 ALDH1B1 Aldehyde dehydrogenase 1 family,member B1 GAACCTTAATGACCAAA 0.23 0.45 AUH AU RNA binding protein/enoyl-Coenzyme A hydratase ATCCCTCCCCACTGACC 0.23 0.14 COMMD1Copper metabolism (Murr1) domain containing 1 TTATATTTTCTTTTAAG 0.230.33 DOCK9 Dedicator of cytokinesis 9 CTTGACATACCTACCAG 0.23 0.45 DUSP1Dual specificity phosphatase 1 AATTTTTTTTCAATGTA 0.23 0.50 MFN2Mitofusin 2 TGGTAGAGCGTTTTCTC 0.23 0.23 PHF12 PHD finger protein 12CATATAATGTACAGTGT 0.23 0.25 PORIMINPro-oncosis receptor inducing membrane injury gene CATTCATTGGTTGTTCA0.23 0.50 SART2 Squamous cell carcinoma antigen recognized by T cells 2GTGGATGTACAGTTTGT 0.23 0.33 THY28 Thymocyte protein thy28CTACTGGGAACAAGTTT 0.23 0.50 UBE2E2 Ubiquitin-conjugating enzyme E2E 2(UBC4/5 homolog, yeast) AGTCAGCTGGAAAGTCT 0.23 0.43 EPS8Epidermal growth factor receptor pathway substrate 8 TACCAGGAACCATTTAA0.25 0.44 ACAD9 Acyl Coenzyme A dehydrogenase family, member 9TCCAAGCTAAAGCCTTA 0.25 0.18 ACAT2 Acetyl-Coenzyme A acetyltransferase 2(acetoacetyl Coenzyme A thiolase) GAGCCTTGGGTACCCCT 0.25 0.33 BAIAP2BAI1-associated protein 2 CCAATGCAGCTGTGAAC 0.25 0.50 C14orf122Chromosome 14 open reading frame 122 CGGCGCTCCCTTCCTTC 0.25 0.50 C9orf60Chromosome 9 open reading frame 60 TACTAGTTTTAGTTTTC 0.25 0.45 CCND1Cyclin D1 (PRAD1: parathyroid adenomatosis 1) TAATTTGCATTACTCTG 0.250.50 EMP1 Epithelial membrane protein 1 TACACCCGCTCTTCAAG 0.25 0.50ERCC3 Excision repair cross-complementing rodent repair deficiency,complementation group 3 (xeroderma pigmentosum group B complementing)AATGCGTGTACTGTTAC 0.25 0.45 FLJ12438 Hypothetical protein FLJ12438AAGACCCCCGTGGAGCT 0.25 0.33 FLJ14827 Hypothetical protein FLJ14827TGTGCTGTGCTGTGTCT 0.25 0.20 GYS1 Glycogen synthase 1 (muscle)GAACTCAGGCCAGGCTC 0.25 0.50 HCFC1 Host cell factor C1 (VP16-accessoryprotein) CGCGCTGTGGGCAATTG 0.25 0.23 HOXB8 Homeo box B8CACCCCCAGGCTCTGCA 0.25 0.33 KIAA1787 G protein pathway suppressor 2AAGAAGGCACGGGTCGG 0.25 0.33 PPAN Peter pan homolog (Drosophila)TTCTTGTTTTGTTATAT 0.25 0.42 PRNPPrion protein (p27-30) (Creutzfeld-Jakobdisease, Gerstmann-Strausler-Scheinkersyndrome, fatal familial insomnia) GGATTTGGCCTTTTCGA 0.25 0.23 RPLP2Ribosomal protein, large, P2 TCAATGGCCTCTTTGTC 0.25 0.45 SSA2TROVE domain family, member 2 TCAGAGATGAGGGCCGC 0.25 0.33 STXBP2Syntaxin binding protein 2 GGGCTGGACGGCTGCGT 0.25 0.45 TNFRSF25Tumor necrosis factor receptor superfamily, member 25 GTCTTTAGGAAATATTG0.25 0.23 UMPS Uridine monophosphate synthetase(orotate phosphoribosyl transferase and orotidine-5′-decarboxylase)AAGTGAGGAGATGGTTA 0.25 0.50 WBP2 WW domain binding protein 2TGTTCCCTTTGTCTTTC 0.27 0.18 MXI1 MAX interactor 1 GGTTCAAGGCCCTGGCC 0.290.40 FLJ22635 Hypothetical protein FLJ22635 CCTGTAATCCCAGATAC 0.29 0.50LOC55974 Stromal cell protein TGGGAAAAAATATTACA 0.29 0.45 MTAPMethylthioadenosine phosphorylase CATTCAGTTGAGTCCCA 0.29 0.20 MTCBP-1Membrane-type 1 matrix metalloproteinase cytoplasmic tailbinding protein-1 GAACACCGTCCCTCTGC 0.29 0.33 PI4KIIPhosphatidylinositol 4-kinase type II CGTGTTGTTCCTGTGCC 0.29 0.20 PKM2Pyruvate kinase, muscle GTAGGTGAGGTGGTTAA 0.29 0.45 TERF2IPTelomeric repeat binding factor 2, interacting protein ATCTCAAAGATACACAG0.29 0.45 VMP1 Transmembrane protein 49 ATGTTAGGGATGTGGAT 0.29 0.25VTI1B Vesicle transport through interactionwith t-SNAREs homolog 1B (yeast) CACTTGCCCTTAAAAAC 0.30 0.20 ACAS2Acetyl-Coenzyme A synthetase 2 (ADP forming) TGTCCCCTCACTCTGTC 0.30 0.50AKT1 V-akt murine thymoma viral oncogene homolog 1 GGTCCCCTCCCCTCTCA0.30 0.50 AMFR Autocrine motility factor receptor AATACTTTAGGGTGGGG 0.300.45 AMPD3 Adenosine monophosphate deaminase (isoform E)GAAAGCATACCTCAGTG 0.30 0.45 AP4B1Adaptor related protein complex 4, beta 1 subunit AGTATCTGGGATGTGAA 0.300.45 ARPC1B Actin related protein 2/3 complex, subunit 1B, 41 kDaTGGCCCTTTCAATATTT 0.30 0.23 C4orf16 Chromosome 4 open reading frame 16GACAGACATCACTACTG 0.30 0.17 C9orf123 Chromosome 9 open reading frame 123TATTTATTGAAAAAAAA 0.30 0.40 COPG Coatomer protein complex, subunit gammaGACTCTCTCAGCTTCCC 0.30 0.50 CTNNBL1 Catenin, beta like 1TCTCTGTGTAGTTCCAG 0.30 0.23 CXADRCoxsackie virus and adenovirus receptor TAAACAGGTTCCTTTGC 0.30 0.45CXorf40 Chromosome X open reading frame 40 GAGCCTGTAAATGTTTT 0.30 0.33DKFZp762N1910 Hypothetical protein DKFZp762N1910 CAAGCCAAAAATATACC 0.300.14 FLJ10379 Hypothetical protein FLJ10379 GACGGGGTGGAGATGGA 0.30 0.45FLJ10404 Hypothetical protein FLJ10404 AACGGGCCGGCGGACGG 0.30 0.45FLJ90652 Hypothetical protein FLJ90652 AATGGCATTGATGCTAA 0.30 0.33FN3KRP Fructosamine-3-kinase-related protein TGTGACATCCGGAGTCC 0.30 0.45FSTL3 Follistatin-like 3 (secreted glycoprotein) TCTAGAGTTCTGCTGGA 0.300.20 FXC1 Fracture callus 1 homolog (rat) TCAAACTGCTTTATTAC 0.30 0.30GNB1 Guanine nucleotide binding protein (G protein), beta polypeptide 1TTAAACCCACCAAAATA 0.30 0.45 HDHD2 Haloacid dehalogenase-like hydrolasedomain containing 2 GTGTGCTGGCTTAAAAT 0.30 0.45 IFNGR2Interferon gamma receptor 2 (interferon gamma transducer 1)ATTTTTGGTGGAATGTT 0.30 0.50 KIAA0436 Prolyl endopeptidase-likeTGTTTCATTCTGATCTT 0.30 0.33 KIAA1838 KIAA1838 TCAAAAACTTGGAGTCA 0.300.33 LRRC5 Leucine rich repeat containing 8 family, member DCCAGCCCTACTGCCGAT 0.30 0.45 MYOIC Myosin IC GGGAGCCGAGTCTTCTG 0.30 0.23NUP188 Nucleoporin 188 kDa TCAACTGGTTCCGGCGT 0.30 0.50 PCK2Phosphoenolpyruvate carboxykinase 2 (mitochondrial) GTGGTGGGCACCTGTAA0.30 0.50 PHF6 PHD finger protein 6 TTTTTTGAAAGCACTGG 0.30 0.50 PLSCR1Phospholipid scramblase 1 GGGAGTAATAGGACCAG 0.30 0.33 PTPRAProtein tyrosine phosphatase, receptor type, A TGGGGCCTGGGTGGGCA 0.300.45 RAB3IL1 RAB3A interacting protein (rabin3)-like 1 TCCACTCAGTAACAAGT0.30 0.45 RABEP1 Rabaptin, RAB GTPase binding effector protein 1GTTGTATAATATTTCAT 0.30 0.50 RNMT RNA (guanine-7-) methyltransferaseCTTAAAAACGCAGAGAG 0.30 0.33 RPL17 Ribosomal protein L17TTTAAACTTTGTGCCTT 0.30 0.50 SHC1 SHC (Src homology 2 domaincontaining) transforming protein 1 TTTTTTATAATAAAACA 0.30 0.30 SLC9A3Solute carrier family 9 (sodium/hydrogen exchanger), isoform 3CTAAAAAATGTAGAAGA 0.30 0.45 SPCS3 Signal peptidase complex subunit 3homolog (S. cerevisiae) ATATTGGGAACCATCTC 0.30 0.50 TGIFTGFB-induced factor (TALE family homeobox) ATTAGGCCTGATTATCT 0.30 0.14TXNRD1 Thioredoxin reductase 1 TTCTTGCTTAAGCCATT 0.30 0.50 UBE2L6Ubiquitin-conjugating enzyme E2L 6 ATGGTTACACTTTTGGT 0.30 0.33 UTXUbiquitously transcribed tetratricopeptide repeat, X chromosomeCTGTTACTGTACTTATG 0.30 0.45 WTAP Wilms tumor 1 associated proteinGGCCCCCCTCCTGGGAT 0.30 0.30 ZNF609 Zinc finger protein 609CCCGTGAGCGAGCTGAC 0.33 0.45 ARF5 ADP-ribosylation factor 5TGAAATCTGATTTTTAT 0.33 0.33 ATP5L ATP synthase, H+ transporting,mitochondrial F0 complex, subunit g GAGTTCGACCTGGGAGC 0.33 0.17 C19orf33Hypothetical LOC541469 protein GCCCCGCCCTCCCCGCG 0.33 0.25 C19orf6Chromosome 19 open reading frame 6 CCATTCTCTTTCAGCTG 0.33 0.50 C6orf111Chromosome 6 open reading frame 111 AGCCACCGTGCCTGGCC 0.33 0.45 CBX5Chromobox homolog 5 (HP1 alpha homolog, Drosophila) GACCCCAAGGCCGCCGA0.33 0.50 CCND1 Cyclin D1 (PRAD1: parathyroid adenomatosis 1)ACATCCCAGAAGAGGAC 0.33 0.40 CORO1C Coronin, actin binding protein, 1CCATTAAAGGGTCTATTA 0.33 0.45 CTL2 CTL2 protein GAGGCCGCTGACTACCG 0.330.50 CTTN Cortactin TGCACTTCACCGCCCTG 0.33 0.50 DCPSDecapping enzyme, scavenger AGGTACTACTACAAACG 0.33 0.23 ELF3E74-like factor 3 (ets domain transcription factor, epithelial-specific)TAAAAACCCAGGGTTCT 0.33 0.30 FLJ20625 Hypothetical protein FLJ20625TTTAATACATAGGTGAT 0.33 0.40 FLJ22875 Hypothetical protein FLJ22875AATGGCACTTAAAATAA 0.33 0.45 FOXJ3 Forkhead box J3 CCCACTGAATTCAGGTC 0.330.50 G3BP2 Ras-GTPase activating protein SH3 domain-binding protein 2ACCCCAGCAACTGTGGT 0.33 0.33 GNS Glucosamine (N-acetyl)-6-sulfatase(Sanfilippo disease IIID) GTGGAGGTTCACAACAA 0.33 0.50 GTPBP6GTP binding protein 6 (putative) AAGTTCCAGAACCAGAA 0.33 0.50 HCAP-GChromosome condensation protein G GAAGTGGCAGTGAAAAA 0.33 0.40 HMGCR3-hydroxy-3-methylglutaryl-Co enzyme A reductase GAAGAAGTAGACTAATC 0.330.50 HSPCA Heat shock 90 kDa protein 1, alpha CCTGGCAGTTGTACTAC 0.330.50 JAGN1 Jagunal homolog 1 (Drosophila) GGTCCAGGGCCTGACAC 0.33 0.45JUP Junction plakoglobin TGTTCAGTTGTGGACCT 0.33 0.33 KIAA1423 KIAA1423GCTGCTCATCCATTACT 0.33 0.50 LOC92345 Hypothetical protein BC008207CTTTGTTTAATGGATTT 0.33 0.50 LOC92912 Hypothetical protein LOC92912TTAAATTCTTAAATGCC 0.33 0.50 MAL2 Mal, T-cell differentiation protein 2TGTAAGAAAAGGCCCAT 0.33 0.40 MCM6 MCM6 minichromosome maintenancedeficient 6 (MIS5 homolog, S. pombe) (S. cerevisiae) TCCAGGCTCTGGTGGGG0.33 0.40 MGC19604 Similar to RIKEN cDNA B230118G17 geneCTTATTGTCCCAATATC 0.33 0.50 MRPL30 Mitochondrial ribosomal protein L30GTTTACCCGCAGACCTT 0.33 0.33 MRPS30 Mitochondrial ribosomal protein S30TACAAACCTGGATTTTT 0.33 0.30 MT1F Metallothionein 1F (functional)CAGGGAATGCCAGTCCG 0.33 0.50 MTA3 Metastasis associated 1 family, member3 CTCTGCCCTCCCTTCTG 0.33 0.25 MYH14 Myosin, heavy polypeptide 14CCCACAATCCCTTTCTA 0.33 0.33 NBS1 Nibrin ACTGCTCATTGTAGATG 0.33 0.50 NCE2NEDD8-conjugating enzyme GAAGACGGTGAAATTGA 0.33 0.30 NCL NucleolinGTCCCAAAATGTCATTG 0.33 0.38 NCOA4 Nuclear receptor coactivator 4TCTACTGTTAGGTGAGG 0.33 0.30 NDRG3 NDRG family member 3 TGATAGTCAGTTGTACA0.33 0.30 PARG1 Rho GTPase activating protein 29 CTTATTTGTTTTAAAAC 0.330.50 PLS3 Plastin 3 (T isoform) AAGGGTAACCATCATCG 0.33 0.45 PSMA4Proteasome (prosome, macropain) subunit, alpha type, 4 AATGGGGGTTATGGGGT0.33 0.30 RAB35 RAB35, member RAS oncogene family GGAGGACGAAGCAGTGG 0.330.30 RALBP1 RalA binding protein 1 AGAAAATTCATAAAGGG 0.33 0.50 RCL1RNA terminal phosphate cyclase-like 1 CCCACCAGGAGCAAGCT 0.33 0.50 RPL4Mitogen activated protein kinase kinase kinase 13 GCCTCTGTCTCCGAGCT 0.330.25 RPLP1 Ribosomal protein, large, P1 GGATTTGGCCTTTTTGC 0.33 0.50RPLP2 Ribosomal protein, large, P2 AAGTTTGTGGATGGCCT 0.33 0.25 RPS3Ribosomal protein S3 AATTCTGAAAGCAAGCC 0.33 0.50 RSBN1LRound spermatid basic protein 1-like GTTGGTCCCTGCGGTGG 0.33 0.50 RTEL1Regulator of telomere elongation helicase 1 AAATGCCACACACATAG 0.33 0.30RTN4 Reticulon 4 TCTGTGCTCAGGAAGAG 0.33 0.40 RUTBC3RUN and TBC1 domain containing 3 CCCTGTAATAAAATTAG 0.33 0.50 SEPN1Selenoprotein N, 1 AATTTGTGAAGGTGGAA 0.33 0.45 SLC25A13Solute carrier family 25, member 13 (citrin) TCCAAGTTCCGTCTTCT 0.33 0.30SLC27A4 Solute carrier family 27 (fatty acid transporter), member 4GCATTTAGTTCAGAGTG 0.33 0.25 SNX11 Sorting nexin 11 AGAAGGATGCTTATTTT0.33 0.46 SPTLC1 Serine palmitoyltransferase, long chain base subunit 1GATTTAAAAATCAAGTT 0.33 0.17 TNPO1 Transportin 1 TTGGAACTCAGACCAGG 0.330.50 TRA16 TR4 orphan receptor associated protein TRA16TGCCTATAGTCCCAGCT 0.33 0.33 TTC8 Tetratricopeptide repeat domain 8AAGTTTCTGATATCTCC 0.33 0.45 USP8 Ubiquitin specific protease 8CTGGCCCGGAGAAGGAA 0.33 0.15 VASP Vasodilator-stimulated phosphoproteinACCAATGTGTCCTAGAA 0.33 0.33 WHSC2 Wolf-Hirschhorn syndrome candidate 2ATTCTTCGGACTGACTG 0.33 0.30 WIPI-2 WIPI49-like protein 2ATGAAACCCTGTCTCTA 0.33 0.20 WSB1 WD repeat and SOCS box-containing 1GATTTAAAAAAAAAAAA 0.33 0.33 WSB1 WD repeat and SOCS box-containing 1TTCCCTGGGAAGACGGG 0.33 0.45 ZBTB4 Zinc finger and BTB domain containing4 CTGGGTGCCCCAGCCTG 0.33 0.50 ZNF335 Zinc finger protein 335TCATCTGTGAATAAAGT 0.33 0.50 ZNF623 Zinc finger protein 623GGTACTCGATGTGTAAT 0.35 0.50 ASPH Aspartate beta-hydroxylaseTTTCGTAGATGGGGTTT 0.38 0.50 C6orf109 Chromosome 6 open reading frame 109GAAGGCAAGATTGTGTC 0.38 0.50 F12 Coagulation factor XII (Hageman factor)CTCACAAGTTTTGGGAA 0.38 0.25 NUP160 Nucleoporin 160 kDa ATTGGTACCCTGACTGC0.38 0.50 SLC25A3 Solute carrier family 25 (mitochondrialcarrier; phosphate carrier), member 3 AAGGCCGAGTAACTGGA 0.38 0.50DKFZP564J0123 Nuclear protein E3-3 GCCCTGACCACAGGGGG 0.38 0.33 EML2Echinoderm microtubule associated protein like 2 CAGGGAAGCCACCAGCT 0.380.50 LLGL2 Lethal giant larvae homolog 2 (Drosophila) TTGAACAAAGTTAAGTC0.40 0.23 C10orf26 Chromosome 10 open reading frame 26 GAGCCCCCGTGATTAGT0.40 0.38 CDC42BPB CDC42 binding protein kinase beta (DMPK-like)TAAATACAAATTTTGTA 0.40 0.40 F2RL1 Coagulation factor II (thrombin)receptor-like 1 CGGTTTAATTGTGGGAG 0.40 0.30 FASN Fatty acid synthaseGGGCTCCAGGAAGCCTG 0.40 0.50 FBXO21 F-box protein 21 TTCACAGTGCAGCTCCT0.40 0.33 FLJ10420 Adaptin-ear-binding coat associated protein 2GCTGCTGCCTGGGCCTC 0.40 0.45 HRIHFB2122 Tara-like proteinCACGTTCCCTAGATGCA 0.40 0.33 IHPK1 Inositol hexaphosphate kinase 1CTGCAGGGCCAAAAGGA 0.40 0.28 K-ALPHA-1 Dehydrin (ERD10) AGCACTGTACTTCATAA0.40 0.43 LEREPO4 Likely ortholog of mouse immediateearly response, erythropoietin 4 CCAAGGGTCCAGGCTGC 0.40 0.45 LOC283680Hypothetical protein LOC283680 ATAGACGCAATGCATTG 0.40 0.38 MORF4L1Mortality factor 4 like 1 ATCGAACAAACCTGAAA 0.40 0.20 MRPL35Mitochondrial ribosomal protein L35 GCGAAACCCTGTCTCTA 0.40 0.30 OTOP2Otopetrin 2 GCTGACGGAAATCTCTT 0.40 0.25 PCYT2Phosphate cytidylyltransferase 2, ethanolamine GTACTGTCTCCACAGCC 0.400.50 PIP5K1C Phosphatidylinositol-4-phosphate 5- kinase, type I, gammaTCTTCTTCGAAGTGGCT 0.40 0.23 PROCR Protein C receptor, endothelial (EPCR)GTTAATTGCTAGTTGGT 0.40 0.14 SELS Selenoprotein S AAGGGAGGGTCCCTGTG 0.400.25 SQSTM1 Sequestosome 1 TAAACGTGGCAGCCAGC 0.40 0.38 TD-60Regulator of chromosome condensation 2 ACAGCGTCTGCTTGCGT 0.40 0.20ZDHHC8 Zinc finger, DHHC-type containing 8 TGGTACTTCTCTTTTCC 0.41 0.45ZDHHC6 Zinc finger, DHHC-type containing 6 CAAGGGCCAAGCAAAGG 0.42 0.50RGL2 Ral guanine nucleotide dissociation stimulator-like 2TGTCTGATGCTGCTGAG 0.42 0.33 RPL28 Ribosomal protein L28TTTGCGGTCCGGGAGGA 0.42 0.20 THG-1 TSC22 domain family, member 4GTGAAACCCCATCTCTA 0.43 0.50 CREB1 CAMP responsive element bindingprotein 1 CTACCCGGTATGACTGG 0.43 0.50 EDG4 Endothelial differentiation,lysophosphatidic acid G-protein-coupled receptor, 4 TGGACCAGGCGCCCAGC0.43 0.29 GPR108 G protein-coupled receptor 108 GTTCCTCAGCCAGGTGG 0.430.30 NUDT14 Nudix (nucleoside diphosphate linked moiety X)-type motif 14AATAAAAGTGGATTTCA 0.43 0.33 PLCB3 Phospholipase C, beta 3(phosphatidylinositol-specific) CGTGCTGGCCACGGCTT 0.43 0.18 RRASRelated RAS viral (r-ras) oncogene homolog CCTGCACACTCCTCCCC 0.43 0.40SPPL2B Signal peptide peptidase-like 2B AATTCAGTGAACTCTTT 0.43 0.50TCTEL1 T-complex-associated-testis-expressed 1-like 1 TGAAACTCATCTCATTA0.43 0.14 TPM3 Tropomyosin 3 TTACTTCAACTAAAAGT 0.44 0.50 FLJ32421Chromosome 1 open reading frame 58 CTGAACTGGATCGTAGG 0.45 0.45 ADPLAdaptor protein containing pH domain,PTB domain and leucine zipper motif 1 AATTTAGAGCATTCCAC 0.45 0.40 ARL10CADP-ribosylation factor-like 10C GCGCAGACTTCCAAATA 0.45 0.30 C10orf9Chromosome 10 open reading frame 9 TTATATACTTTTCAGTA 0.45 0.45 Cl4orf1Chromosome 14 open reading frame 1 TTTCACCCCTTTTCTTC 0.45 0.18 C14orf92Chromosome 14 open reading frame 92 TACAAATAATAAAATGT 0.45 0.45 C6orf4TRAF3 interacting protein 2 TGAAGGTGGATTGGTCG 0.45 0.45 CABIN1Calcineurin binding protein 1 GCCGTGAACTTTATGCT 0.45 0.45 CARD15Caspase recruitment domain family, member 15 TTTGATTTTAGTAGTAT 0.45 0.50CCAR1 Cell division cycle and apoptosis regulator 1 TTGGTCAGGCTGGTCTG0.45 0.45 CDC42 Cell division cycle 42 (GTP binding protein, 25 kDa)GAGCAATTCTAGGGGCT 0.45 0.50 CDC42SE1 CDC42 small effector 1CAGATAAACTTCTTCAG 0.45 0.50 CNOT10 CCR4-NOT transcription complex,subunit 10 TTGCCTCCTGAGCAAAG 0.45 0.33 CSTF2Cleavage stimulation factor, 3′ pre-RNA, subunit 2, 64 kDaGTCTCTTTGGGCGGAAG 0.45 0.45 CYCS Cytochrome c, somatic AACCTGAACAAAGAAAG0.45 0.45 DBR1 Debranching enzyme homolog 1 (S. cerevisiae)GTGCTGTTTAATTGTAA 0.45 0.45 DC-UbP Dendritic cell-derived ubiquitin likeprotein CATTGTTGGCTATTTGA 0.45 0.45 DDX26DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 26 CCAGGGAGATCTTTGAC 0.450.17 ENO1 Enolase 1, (alpha) TCAATTCATAAAAACAA 0.45 0.50 EREG EpiregulinAAATCCTAGAATGTATG 0.45 0.50 FAM44B Family with sequence similarity 44,member B GTTTGTTGGGAAGGTAA 0.45 0.50 FBXO28 F-box protein 28TGGCGGAGTACCAAGAC 0.45 0.50 FLJ20558 Hypothetical protein FLJ20558CGTTGTCTGCCCACCCC 0.45 0.45 FLJ33817 Hypothetical protein FLJ33817TGTAAGATGCACAGTAT 0.45 0.45 GOPC Golgi associated PDZ and coiled-coilmotif containing CTCGGATTCAAGCAGCT 0.45 0.45 GSK3BGlycogen synthase kinase 3 beta CCACCACAAAGGCCCTT 0.45 0.30 H2AFXH2A histone family, member X TCCCCCGTGCACGGTTC 0.45 0.38 HOXB6Homeo box B6 GTTCTCTTTGTACGATA 0.45 0.50 IDSIduronate 2-sulfatase (Hunter syndrome) GAGTGAAATTCTTGTTT 0.45 0.33KLHL12 Kelch-like 12 (Drosophila) TAAAACAATGTAATTGA 0.45 0.50 LOC286148Hypothetical protein LOC203107 TGGATCACCAAGATACA 0.45 0.50 LOC400027Hypothetical gene supported by BC047417 CTTTGCTGTATGTCTTC 0.45 0.45 LSM5LSM5 homolog, U6 small nuclear RNA associated (S. cerevisiae)TTTTTAGGTGACTTTTT 0.45 0.50 MARCH-VI Membrane associated ring finger(C3HC4) 6 CCTGTGGTTTTGTGTTT 0.45 0.50 MARK3MAP/microtubule affinity-regulating kinase 3 CCCTTCACTTAACTAGG 0.45 0.50MCCC2 Methylcrotonoyl-Coenzyme A carboxylase 2 (beta) TCCTGCTGCCGGCAAAA0.45 0.20 MGC13114 Hypothetical protein MGC13114 TACTTACAAAAACTGAG 0.450.23 MGC14156 Hypothetical protein MGC14156 GGCTGTAAGTTGTACTT 0.45 0.33MGC3262 Hypothetical protein MGC3262 TTAGCCAGGCTGGTCTT 0.45 0.45 MRPL33Mitochondrial ribosomal protein L33 TAAATTATTTCATATAT 0.45 0.25 MTMR6Myotubularin related protein 6 GATCAAAATTTGTGTAA 0.45 0.25 MYO1BMyosin IB TTTTCCAAAATGTTTTT 0.45 0.20 NAP1L160S ribosomal protein L6 (RPL6A) GTGGCATAGCATCTGAG 0.45 0.33 NCOA6Nuclear receptor coactivator 6 TCACTGATGGTCAGATT 0.45 0.50 NEK6NIMA (never in mitosis gene a)-related kinase 6 AAATAAAAAATAAAAAT 0.450.45 NFIC Nuclear factor I/C (CCAAT-binding transcription factor)CAGAGGCCCTCAAGTGA 0.45 0.45 NFKBIL1Nuclear factor of kappa light polypeptidegene enhancer in B-cells inhibitor-like 1 GCTCCGGTGTCCGGCTC 0.45 0.45NKAP NF-kappaB activating protein TAGTAAAGACATCTTAT 0.45 0.50 NUDT4P1Nudix (nucleoside diphosphate linked moiety X)-type motif 4 pseudogene 1ATTTATAATTTCACTGA 0.45 0.45 ORC4L Origin recognition complex, subunit 4-like (yeast) ATGTATGGGGATTAGAA 0.45 0.50 PLIPProtein tyrosine phosphatase, mitochondrial 1 TTGTCCCTGGCAAACCT 0.450.50 PNPLA4 Patatin-like phospholipase domain containing 4ATCAGTTAAGTCACTCT 0.45 0.45 PRDX3 Peroxiredoxin 3 GTTTTTAAATAAGATTA 0.450.45 PREI3 Preimplantation protein 3 ATTCTGGTGGAGATTCC 0.45 0.50 PRSS16Protease, serine, 16 (thymus) GCCCTGAAACACACACA 0.45 0.23 RAB5BRAB5B, member RAS oncogene family GCAATATGTATTTCCCT 0.45 0.45 RAB6ARAB6A, member RAS oncogene family ATAGGATTGCCTAGTGT 0.45 0.45 RAB7L1RAB7, member RAS oncogene family- like 1 GCGAGAATCCAGCTTTG 0.45 0.23RAD9A RAD9 homolog A (S. pombe) CTTTTCTGAAGAGCCGG 0.45 0.45 RHOBRas homolog gene family, member B AATGGATTACCAACAAA 0.45 0.50 RPL17Ribosomal protein L17 GACAAGATCTATGAAGG 0.45 0.50 RPL5Ribosomal protein L5 TGTTCATCATCTTAAGT 0.45 0.50 RTN4 Reticulon 4CAGCGCACAGATGTGCT 0.45 0.45 SAMD1Sterile alpha motif domain containing 1 TCTTTTCTTGTCATCCT 0.45 0.50SFXN1 Sideroflexin 1 CACTCAGTGTGGACTGG 0.45 0.45 SLC1A3Solute carrier family 1 (glial highaffinity glutamate transporter), member 3 ACCAGGTCCACTGTGGA 0.45 0.50SLC5A6 Solute carrier family 5 (sodium-dependent vitamin transporter), member 6 GGACTGGGTCGTCTGAA 0.45 0.45SNAP29 Synaptosomal-associated protein, 29 kDa GGTTGTATTTTTCTGGT 0.450.25 STX7 Syntaxin 7 AAGAAGTGAGCTTAGTT 0.45 0.33 TAF1BTATA box binding protein (TBP)- associated factor, RNA polymerase I, B,63 kDa GTGATGCGCATAGGCCT 0.45 0.30 TCIRG1T-cell, immune regulator 1, ATPase, H+transporting, lysosomal V0 protein a isoform 3 CCACTGCACTCCAGACT 0.450.50 TMC2 Transmembrane channel-like 2 GAGGGCCTTGTGGACAC 0.45 0.25 TSC2Tuberous sclerosis 2 TATGTCAACTCATTACT 0.45 0.30 UBE2D1Ubiquitin-conjugating enzyme E2D 1 homolog, yeast) TTGATCCTCTTGCAAGC0.45 0.30 UBR2 Ubiquitin protein ligase E3 component n-recognin 2TAGGAGAATCCAAGCGA 0.45 0.45 VDR Vitamin D (1,25- dihydroxyvitamin D3)receptor AATATTTCAGTGCTGCT 0.45 0.45 ZBTB34Zinc finger and BTB domain containing 34 AGTACCTATTTATGTGG 0.45 0.45ZCCHC6 Zinc finger, CCHC domain containing 6 TAAATGTTAACAATTAG 0.45 0.45ZDHHC2 Zinc finger, DHHC-type containing 2 GTCTGCCAGCCTGGCTC 0.45 0.50ZFPM1 Zinc finger protein, multitype 1 AGGGGGCTGAGAGGTTT 0.45 0.33ZFYVE27 Zinc finger, FYVE domain containing 27 TCACCGGTCAGTGCCTT 0.450.15 GSN Gelsolin (amyloidosis, Finnish type) CAGGGGAGTGGGCCCGG 0.450.50 MPG N-methylpurine-DNA glycosylase CCTCCCTGATGGGTGGG 0.46 0.50 DNM2Dynamin 2 GTACTGTAGCAGGGGAA 0.47 0.25 ITGA3Integrin, alpha 3 (antigen CD49C, alpha 3 subunit of VLA-3 receptor)ACCTGCCCCTCTTTACT 0.50 0.50 AAASAchalasia, adrenocortical insufficiency, alacrimia (Allgrove, triple-A)AAGGTGGAGTGTGACCT 0.50 0.17 ABCF1 ATP-binding cassette, sub-family F(GCN20), member 1 TTGGTAAGGCTGATCTC 0.50 0.25 ACBD5Acyl-Coenzyme A binding domain containing 5 GTCCTAGATTGTGGATA 0.50 0.33ACTR10 Actin-related protein 10 homolog (S. cerevisiae)GTGGCGCACACCTGTAA 0.50 0.45 ADAT1 Adenosine deaminase, tRNA-specific 1CTTCTGAAAACAACAGG 0.50 0.25 ADK Adenosine kinase TATTTGTTGAGTTTTGC 0.500.33 ADMP Likely ortholog of androgen downregulated gene expressed in mouse prostate GTGGCTTACACCTGTAA 0.50 0.45APG12L APG12 autophagy 12-like (S. cerevisiae) AAAGTGGAAACATTGGT 0.500.25 APG5L APG5 autophagy 5-like (S. cerevisiae) CCACTGCACTCCAGTCT 0.500.25 APOL6 Apolipoprotein L, 6 GCAATGAAAATTTTAAG 0.50 0.50 APRINAndrogen-induced proliferation inhibitor TATTTTTACTGATCACA 0.50 0.33ARHGEF12 Rho guanine nucleotide exchange factor (GEF) 12GGAAAAAAAAATCCTGT 0.50 0.33 ATP5E ATP synthase, H+ transporting,mitochondrial F1 complex, epsilon subunit TTTGCCTGTTAAGTTGT 0.50 0.33ATP6V1H ATPase, H+ transporting, lysosomal 50/57 kDa, V1 subunit HCTAAAGTACTTTAACTG 0.50 0.45 BBX Bobby sox homolog (Drosophila)CTGAAAATTGCTGAGAT 0.50 0.45 BYSL Bystin-like TTCTGAAAGGATTCACT 0.50 0.23C10orfl37 Chromosome 10 open reading frame 137 AAGAAGCAGGGCCTCTA 0.500.18 C1orf8 Chromosome 1 open reading frame 8 AGCAAGAAACTGCCTGC 0.500.48 C3orf4 Chromosome 3 open reading frame 4 TATTAGAGAATGAAAAG 0.500.33 C6orf55 Chromosome 6 open reading frame 55 TCAAGAGCCGAAGGAAT 0.500.50 C6orf66 Chromosome 6 open reading frame 66 CCCCAGTTGCTGATCAA 0.500.50 CAPNS1 Calpain, small subunit 1 TGCTAATCAAACCTGCT 0.50 0.50 CBR4Carbonic reductase 4 TGTTTAATACAAGTTAA 0.50 0.50 CCNL2 Cyclin L2GTCCAGAATGATGTTTG 0.50 0.50 CDC23 CDC23 (cell division cycle 23, yeast,homolog) GCTCGGCCGCTAGTGCC 0.50 0.33 CDC34 Cell division cycle 34AAAGTGGGTGGAGCCCA 0.50 0.45 CDC42 Cell division cycle 42 (GTP bindingprotein, 25 kDa) TAAGGTATTGCAAATAA 0.50 0.45 CDCA7Cell division cycle associated 7 TTACCGTCCCCTACCTC 0.50 0.33 CHD3Chromodomain helicase DNA binding protein 3 TCCAAAGCATTGACTGT 0.50 0.33CHP Calcium binding protein P22 TCTTGTCATACAAATTT 0.50 0.45 CHUKConserved helix-loop-helix ubiquitous kinase TCAACACAGATCGAGAA 0.50 0.33CIR CBF1 interacting corepressor CCTGTGGTCCCAGCTAC 0.50 0.45 CLN8Ceroid-lipofuscinosis, neuronal 8 (epilepsy, progressive with mentalretardation) CGGGAGACATCTTTGGC 0.50 0.33 CLTAClathrin, light polypeptide (Lca) CGGGAGCACCCGGCGCT 0.50 0.14 COMTD1Catechol-O-methyltransferase domain containing 1 CTCCCTTGCCCTGACAT 0.500.50 COPZ1 Coatomer protein complex, subunit zeta 1 TGTCTGGATGAAGCTGG0.50 0.50 CYB561D2 Cytochrome b-561 domain containing 2TAGACAATGCTGCTAAG 0.50 0.25 D15Wsu75e DNA segment, Chr 15, Wayne StateUniversity 75, expressed TACAGAACACACAATTT 0.50 0.50 DAZAP2DAZ associated protein 2 ACTATAGAGACCCCGTG 0.50 0.33 DDX11DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 11 (CHL1-like helicasehomolog, S. cerevisiae) AAGGCCCCTGCCGCCAT 0.50 0.50 DKFZP564K1964DKFZP564K1964 protein AATATGGGTGATTTTGA 0.50 0.33 DNAJC7DnaJ (Hsp40) homolog, subfamily C, member 7 GGCTCACTTTAAAAAAA 0.50 0.10DUSP6 Dual specificity phosphatase 6 CCTGTGGCCAAGCTGGC 0.50 0.45EIF3S6IP Eukaryotic translation initiation factor 3,subunit 6 interacting protein AATGTGGCTGACCTTAT 0.50 0.38 EIF4A2Eukaryotic translation initiation factor 4A, isoform 2 GTCGGGGCGTCCACGCC0.50 0.50 ERP70 Protein disulfide isomerase family A, member 4CAGCAGATAATTGTTCA 0.50 0.45 ESCO1 Establishment of cohesion 1 homolog 1(S. cerevisiae) AAAAAACTCCAAATAAG 0.50 0.45 ESDEsterase D/formylglutathione hydrolase AGCAGTGACGGATAGTT 0.50 0.50 EVA1Epithelial V-like antigen 1 AAACTAACATTCCAAGG 0.50 0.33 FAIMFas apoptotic inhibitory molecule AGGGTGTCTTCATTTGT 0.50 0.45 FBLFibrillarin TGAATGTGGGTGAGTTT 0.50 0.30 FBXO31 F-box protein 31TGATTGATTTGTAATTT 0.50 0.25 FBXO7 F-box protein 7 TAAACGGCCTCATTTCT 0.500.50 FEM1A Fem-1 homolog a (C.elegans) CACACCAGTTACTTCCT 0.50 0.25FLJ10099 Hypothetical protein FLJ10099 TAAGCAGCACGTTTTAA 0.50 0.45FLJ12892 Coiled-coil domain containing 14 CTGCCCTCGGCCTGTTC 0.50 0.17FLJ13909 Hypothetical protein FLJ13909 CACACAGCACAATTCAG 0.50 0.45FLJ14775 Hypothetical protein FLJ14775 TTTGAAAATTTAATTAA 0.50 0.40FLJ20397 Hypothetical protein FLJ20397 CCTGCTGAGGAGTTCAG 0.50 0.50FLJ21148 Hypothetical protein FLJ21148 ACGTCGTCGACCTTGGC 0.50 0.45FLJ36878 Hypothetical protein FLJ36878 AGCCACTGCACCTGGCC 0.50 0.50FLJ38819 Harmonin-interacting ankyrin repeat containing proteinGATCTTTTGTCCTCACT 0.50 0.30 FLN29 TRAF-type zinc finger domaincontaining 1 GGCGTTTAGAGTTATAC 0.50 0.23 FSCN1Fascin homolog 1, actin-bundling protein (Strongylocentrotus purpuratus)AACTGGGCACCTCCGGG 0.50 0.45 GALNS Galactosamine (N-acetyl)-6-sulfatesulfatase (Morquio syndrome, mucopolysaccharidosis type IVA)TACTTGCTATATTGAGG 0.50 0.33 GALNT2 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- acetylgalactosaminyltransferase 2(GalNAc-T2) TACCAGCTCTTCCGCAG 0.50 0.45 GSK3AGlycogen synthase kinase 3 alpha AACTTACAGAATTAAAG 0.50 0.15 GTL3Gene trap locus 3 (mouse) TGCCTGTGGTCCCAGCT 0.50 0.45 HMFN0672Hypothetical gene supported by AK129923 GACTATGGGGGTGCCGG 0.50 0.33HOXA3 Homeo box A3 GATTTCTACTGAGTTGG 0.50 0.33 HSD17B7Hydroxysteroid (17-beta) dehydrogenase 7 CTAAACTTTTTATAAAA 0.50 0.47 ID2Inhibitor of DNA binding 2, dominant negative helix-loop-helix proteinGCAAAACCCTGTCTCTA 0.50 0.33 IKBKBInhibitor of kappa light polypeptide geneenhancer in B-cells, kinase beta TCACCTTAGGTAGTAGG 0.50 0.08 ITM2BIntegral membrane protein 2B ACACAGTATTCGCTCTT 0.50 0.50 ITRIntimal thickness-related receptor GCTGTTGGTGGGACCCG 0.50 0.50 JAG2Jagged 2 CTGATGCCCAAGGGCAA 0.50 0.30 KIAA0063 KIAA0063 gene productTGTTAATTTATTGAGTG 0.50 0.40 KIAA0194 KIAA0194 protein AAAGGAATGAGCCCTAG0.50 0.33 KIAA0652 KIAA0652 gene product CTGCTAAGGTAGTGAAT 0.50 0.25KIAA0746 KIAA0746 protein TATCCCAGAACTTAAAG 0.50 0.29 KIAA1229KIAA1229 protein AGATGAGATGACCACCA 0.50 0.20 KLF6 Kruppel-like factor 6GCCTTGGGTGACAAATT 0.50 0.23 LIF Hypothetical protein MGC20647TAAAGAGTGGTGGACTT 0.50 0.45 LKAP Limkain b1 GATGAAGAGATTAAACC 0.50 0.50LOC119710 Hypothetical protein BC009561 TAAATTACAAGCCCCAG 0.50 0.33LOC124245 Hypothetical protein BC001584 TCAATAAATTCATACTT 0.50 0.50LOC129285 Smooth muscle myosin heavy chain 11 isoform SM1-likeAGTGGATTTTATTTACC 0.50 0.25 LOC139886 Hypothetical protein LOC139886TTCAAAAAGGAATTACA 0.50 0.50 L0055831 30 kDa protein TGCATCGCGGAGCAGCC0.50 0.50 LOC96610 Hypothetical protein similar to KIAA0187 gene productTTTTAGACCTAGAAAAG 0.50 0.30 LRBA LPS-responsive vesicle trafficking,beach and anchor containing GCTAGGAGTCCCAGGGA 0.50 0.50 LSSLanosterol synthase (2,3-oxidosqualene- lanosterol cyclase)AAACAGTAGTGTTCCCA 0.50 0.50 MAP1LC3BMicrotubule-associated protein 1 light chain 3 beta TCTCCTACCCCTCACTG0.50 0.45 MCFP Mitochondrial carrier family protein GTGCTATTATTAGGCTT0.50 0.50 MCL1 Myeloid cell leukemia sequence 1 (BCL2-related)CAGTAGATAGAGGGGAG 0.50 0.45 MGC13024 Hypothetical protein MGC13024GAGAAACCCCGTCTCTA 0.50 0.17 MGC16385 Hypothetical protein MGC16385AGCAATTTCATCAAATC 0.50 0.50 MGC3222 Transmembrane protein 43TCTGCAAGAAGGCCTCC 0.50 0.50 MRPS21 Mitochondrial ribosomal protein S21ACTTGCACAAGATGGCA 0.50 0.50 MRPS7 Mitochondrial ribosomal protein S7CTAGGTATGGATCTCCT 0.50 0.40 MTX2 Metaxin 2 GTGGGGGCAACTCAAAC 0.50 0.23NARS Asparaginyl-tRNA synthetase AAATGACTTATGGGGGA 0.50 0.45 NCOA3Nuclear receptor coactivator 3 CCCCCTGCTCCTGTGCC 0.50 0.15 NONONon-POU domain containing, octamer- binding TCCAGGCAGTGTGAGGA 0.50 0.33PANK2 Pantothenate kinase 2 (Hallervorden- Spatz syndrome)ATAAATATAATCAGTAT 0.50 0.33 PDLIM5 PDZ and LIM domain 5TGCCAGCCTCATTCGAA 0.50 0.45 PEX11B Peroxisomal biogenesis factor 11BTTATGAGAGTTCTGGAG 0.50 0.45 PIK3C3 Phosphoinositide-3-kinase, class 3GGCTGCCGAGTCCTGCC 0.50 0.50 PINX1 PIN2-interacting protein 1GTGTTTATTTTCTTTCT 0.50 0.50 PPP3CA Protein phosphatase 3 (formerly 2B),catalytic subunit, alpha isoform (calcineurin A alpha) CAAGCTGTAACTTCCCT0.50 0.30 PPP4R2 Protein phosphatase 4, regulatory subunit 2GGGAAGTGTGCCCAGCT 0.50 0.45 PRDX1 Peroxiredoxin 1 GAACAGCAAACGCCTGT 0.500.50 PRKAR2B Protein kinase, cAMP-dependent, type II, betaTGTGATCACAAAGACTG 0.50 0.45 PSMB10 Proteasome (prosome, macropain)subunit, beta type, 10 AGGGATCCTATTTGTCT 0.50 0.40 PSMD12Proteasome (prosome, macropain) 26S subunit, non-ATPase, 12AAGAGGGAAGGAAAAGA 0.50 0.40 PSME4 Proteasome (prosome, macropain)activator subunit 4 AGGAGAGAAGACCCTGC 0.50 0.50 PYGO2Pygopus homolog 2 (Drosophila) GTATTAGGTTTTTTGAG 0.50 0.33 RAB10RAB10, member RAS oncogene family TGGGCCTTCCCCAGGAG 0.50 0.25 RBM14RNA binding motif protein 14 AGTTCTTCCAGGGGACC 0.50 0.50 RBMXRNA binding motif protein, X-linked TCCTTACTAGGTGTTTT 0.50 0.33 RGS19Regulator of G-protein signalling 19 CCTCCTATTACTGAAGT 0.50 0.50 RIOK3RIO kinase 3 (yeast) AGCTGTCTGGCCTGTGA 0.50 0.30 RIPRPA interacting protein GTCCCCTCTGGGGCGTC 0.50 0.25 RNF126Ring finger protein 126 TGGTACTACTGAAGAAG 0.50 0.25 RNF14Ring finger protein 14 CTCACCTGCTACAGCCG 0.50 0.33 SEZ6L2Seizure related 6 homolog (mouse)-like 2 GCCCACAGTAGAATATC 0.50 0.25SFRS4 Splicing factor, arginine/serine-rich 4 GACAAGGAAGGCAATGT 0.500.50 SFXN4 Sideroflexin 4 CAGATTTCCAATCAGTG 0.50 0.40 SLC37A3Solute carrier family 37 (glycerol-3- phosphate transporter), member 3CCCCTCCCTCCTTTTTA 0.50 0.50 SLC4A2Solute carrier family 4, anion exchanger, member 2 (erythrocyte membraneprotein band 3-like 1) CTTCTGCAAATTCGAAT 0.50 0.43 SLC7A1Solute carrier family 7 (cationic amino acid transporter, y+system), member 1 GCAGTGGCCTCAGCCTT 0.50 0.29 SLC9A3R1Solute carrier family 9 (sodium/hydrogen exchanger), isoform 3regulator 1 TCCCAGCCCACATAGAT 0.50 0.40 SPR Sepiapterin reductase (7,8-dihydrobiopterin:NADP+ oxidoreductase) CCCTCCATTTGTAAGAA 0.50 0.50SQSTM1 Sequestosome 1 CTGAAACAGCTAGAAAA 0.50 0.45 SUPV3L1Suppressor of var1, 3-like 1 (S. cerevisiae) CTAAAGGAGGTATCTTG 0.50 0.50TCF12 Transcription factor 12 (HTF4, helix-loop-helix transcription factors 4) GTGCACTGTGAACCTGA 0.50 0.25 TEGTTestis enhanced gene transcript (BAX inhibitor 1) ACAATATCGACACCAGT 0.500.33 TPT1 Tumor protein, translationally-controlled 1 GGATTAACTTGAGGGTC0.50 0.20 TRNT1 TRNA nucleotidyl transferase, CCA- adding, 1CTAATTTAACTAGTCAC 0.50 0.25 UBADC1Ubiquitin associated domain containing 1 ATTAATAAAAAAGGCAA 0.50 0.20UBE4B Ubiquitination factor E4B (UFD2 homolog, yeast) TGTCTTTGCTCTTTCTG0.50 0.50 UBQLN1 Ubiquilin 1 ATTTCAATCTGCCAAAG 0.50 0.33 USMG5Upregulated during skeletal muscle growth 5 ATATTAGCAAAGGTAAA 0.50 0.33VAPA VAMP (vesicle-associated membraneprotein)-associated protein A, 33 kDa CAGTCCCGGCTGGCCAC 0.50 0.50 VPS24Vacuolar protein sorting 24 (yeast) GTCTTTCACCCAGCCAG 0.50 0.50 ZDHHC13 Zinc finger, DHHC-type containing 13 TGATGTTTTAGTGCTTT 0.50 0.33 ZIC2Zic family member 2 (odd-paired homolog, Drosophila) TAACAGGAAATTAAATG0.50 0.33 ZMAT2 Zinc finger, matrin type 2 TCTAAAAAGGCACAGAA 0.50 0.50ZNF185 Zinc finger protein 185 (LIM domain) ATGAACACGGTGATGAC 0.50 0.45ZNF403 Zinc finger protein 403 GACTGTCTCATACTGCT 0.50 0.30 ZNF584Zinc finger protein 584

Having described the invention, we claim:
 1. A method of determining thesusceptibility and/or responsiveness of a cancer cancer cells,precancerous cells, and/or benign tumor cells of a subject to treatmentwith an inhibitor of one or more enzymes of a glutamine metabolismpathway of the cancer cells, the precancerous cells, and/or the benigntumor cells, comprising: obtaining a sample of the cancer cells, theprecancerous cells, and/or the benign tumor cells from the subject;assaying the cancer cells, the precancerous cells, and/or the benigntumor cells for the presence a mutated PIK3CA gene or a mutant form ofPIK3CA protein or a biologically active fragment thereof; anddetermining that the subject should be treated with the inhibitor if thecancer cells have the mutated PIK3CA gene or the mutant form of PIK3CAprotein or a biologically active fragment thereof.
 2. The method ofclaim 1, wherein cancer cells and the precancerous cells are obtainedfrom a tumor or biological sample of the subject.
 3. The method of claim1, wherein the mutation is detected using an amplification assay or bymolecular cloning or sequencing or microarray analysis.
 4. The method ofclaim 1, wherein the PIK3CA gene in the sample is amplified bypolymerase chain reaction or ligase chain reaction.
 5. The method ofclaim 1, wherein a DNA hybridization assay is used to detect the PIK3CAgene in the sample.
 6. The method of claim 1, wherein the cancer cellsare selected from the group consisting of lung cancer, digestive andgastrointestinal cancers, gastrointestinal stromal tumors,gastrointestinal carcinoid tumors, colon cancer, rectal cancer, analcancer, bile duct cancer, small intestine cancer, and stomach (gastric)cancer, esophageal cancer, gall bladder cancer, liver cancer, pancreaticcancer, appendix cancer, breast cancer, ovarian cancer, renal cancer,cancer of the central nervous system, skin cancer, lymphomas,choriocarcinomas, head and neck cancers, osteogenic sarcomas, and bloodcancers.
 7. The method of claim 1, wherein the inhibitor comprises atleast one of a glutaminase inhibitor or an aminotrasferase inhibitor. 8.The method of claim 1, wherein the inhibitor comprises at least one ofaminooxyacetate (AOA) or epigallocatechin gallate (EGCG).
 9. The methodof claim 1, wherein the inhibitor comprises at least one ofbis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide or CB-839.10. The method of claim 1, further comprising administering theinhibitor to cancer cells of the subject if the cancer cells have themutated PIK3CA gene or the mutant form of PIK3CA protein or abiologically active fragment thereof.
 11. A method of treating a subjecthaving cancer, precancerous cells, or a benign tumor that has a mutatedPIK3CA gene or protein, the method comprising: administering to thesubject a therapeutically effective amount of an inhibitor of one ormore enzymes of a glutamine metabolism pathway of the cancer cells. 12.The method of claim 11, wherein the inhibitor comprises at least one ofa glutaminase inhibitor or an aminotrasferase inhibitor.
 13. The methodof claim 11, wherein the inhibitor comprises at least one ofaminooxyacetate (AOA) or epigallocatechin gallate (EGCG).
 14. The methodof claim 11, wherein the inhibitor comprises at least one ofbis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide or CB-839.15. The method of claim 11, further comprising determining that thecancer cells have the mutated PIK3CA gene or the mutant form of PIK3CAprotein or a biologically active fragment thereof prior toadministration of the inhibitor.
 16. The method of claim 15, wherein thedetermining step includes: obtaining a sample of cancer cells,precancerous cells, and/or benign tumor cells from the subject; andassaying the cancer cells, the precancerous cells, and/or the benigntumor cells for the presence a mutated PIK3CA gene or a mutant form ofPIK3CA protein or a biologically active fragment thereof.
 17. The methodof claim 16, wherein the PIK3CA gene in the sample is amplified bypolymerase chain reaction or ligase chain reaction.
 18. The method ofclaim 16, wherein a DNA hybridization assay is used to detect the PIK3CAgene in the sample.
 19. A method of treating a subject having cancer,precancerous cells, or a benign tumor that has mutates PIK3CA gene ofprotein, the method comprising determining that the cancer cells havethe mutated PIK3CA gene or the mutant form of PIK3CA protein or abiologically active fragment thereof, and administering to the subject atherapeutically effective amount of an inhibitor of one or more enzymesof a glutamine metabolism pathway of the cancer cells if the cancercells harbor a PIK3CA mutation.
 20. The method of claim 19, wherein thedetermining step includes: obtaining a sample of cancer cells,precancerous cells, and/or benign tumor cells from the subject; andassaying the cancer cells, the precancerous cells, and/or the benigntumor cells for the presence a mutated PIK3CA gene or a mutant form ofPIK3CA protein or a biologically active fragment thereof.